COMPOSITIONS COMPRISING A MODIFIED GlcNAc-1-PHOSPHOTRANSFERASE AND METHODS OF USE THEREOF

ABSTRACT

The disclosure provides a modified UDP-GlcNAc:Lysosomal Enzyme GlcNAc-1-phosphotransferase with enhanced ability to phosphorylate lysosomal enzymes and methods of use thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/402,468, filed Sep. 30, 2016 the disclosure of which is herebyincorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under CA 008759 awardedby the National Institutes of Health (NIH). The government has certainrights in the invention.

FIELD OF THE INVENTION

The disclosure provides a modified UDP-GlcNAc:Lysosomal EnzymeGlcNAc-1-phosphotransferase with enhanced ability to phosphorylatelysosomal enzymes and methods of use thereof.

BACKGROUND OF THE INVENTION

Enzyme Replacement Therapy (ERT) is currently the major form oftreatment for a number of lysosomal storage diseases, although itsefficacy varies among the individual disorders. Most of these inheriteddisorders arise from the lack of activity of a single lysosomal enzymewhich leads to the accumulation of the material normally degraded by theenzyme. The buildup of the storage material in the lysosome eventuallyresults in cell and organ dysfunction. The goal of ERT is to introducesufficient amounts of normal enzyme into the lysosomes of the defectivecells to clear the storage material and restore lysosome function. Thisform of therapy was first used in patients with Type 1 Gaucher diseasewho lack acid β-glucocebrosidase activity and accumulateglucosylceramide primarily in macrophage type cells. The replacementenzyme, containing N-linked glycans with terminal mannose residues, isinfused intravenously and taken up by macrophages via cell surfacemannose receptors. The endocytosed enzyme is then transported viaendosomes to lysosomes where it functions with good clinical results inthis disorder.

Since most cell types lack mannose receptors, the replacement enzymesused to treat lysosomal storage disorders that involve cell types otherthan macrophages utilize binding to mannose 6-phosphate (Man-6-P)receptors at the cell surface for subsequent delivery to lysosomes.These enzymes are purified from the secretions of mammalian cells,mostly Chinese Hamster Ovary cells, engineered to produce high levels ofthe enzyme of interest. This approach is dependent upon the ability ofthe endogenous GlcNAc-1-phosphotransferase to phosphorylate mannoseresidues of the N-glycans of the expressed lysosomal enzyme. Some of thereplacement enzymes produced by this technique are highly phosphorylatedand bind well to the Man-6-P receptors. Others, however, are poorlyphosphorylated, limiting their effectiveness in ERT. This includes thePompe disease enzyme (acid α-glucosidase, GAA) and thealpha-mannosidosis enzyme (lysosomal acid α-mannosidase, LAMAN).

Thus, there is a need in the art for improved methods of enzymereplacement therapy and improved enzyme production.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides a modifiedGlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/β subunit comprisinginternal deletion of amino acids with reference to full-length humanGlcNAc-1-PT α/β subunit of SEQ ID NO:1. The full-length GlcNAc-1-PT α/βincludes a spacer-1 domain (spacer-1), a Notch 1 domain (Notch 1), aNotch 2 domain (Notch 2), a spacer-2 domain (spacer-2), a DNAmethyltransferase-associated protein interaction domain (DMAP), aspacer-3 domain (spacer-3), a α/β subunit cleavage site, and a spacer-4domain (spacer-4), arranged from the N- to C-terminus of thepolypeptide. In the modified GlcNAc-1-PT α/β subunit, the spacer-1 isinternally deleted. In addition, the region between the Notch-1 and α/βsubunit cleavage site may also be deleted.

In another aspect, the disclosure provides a vector comprising thepolynucleotide of a modified GlcNAc-1-PT α/β subunit, in which thespacer-1, or spacer-1 and region between the Notch-1 and α/β subunitcleavage site is deleted.

In an aspect, the disclosure provides a host cell that includes a vectorcomprising the polynucleotide of a modified GlcNAc-1-PT α/β subunit, inwhich the spacer-1, or spacer-1 and region between the Notch-1 and α/βsubunit cleavage site are deleted.

In an aspect, the disclosure provides a method to increaseoligosaccharide phosphorylation of a protein of interest, such asβ-glucocebrosidase (GBA), GalA, Cathepsin D (CathD), Niemann-Pickdisease type C2 (NPC2), β-hexosaminidase (HEXB), α-Galactosidase (GLA),β-Mannosidase (MANBA), alpha-L-idurnoidase, iduronate sulfatase,arylsulfatase B, acid α-glucosidase (GAA), or lysosomal acidα-mannosidase (LAMAN), by expressing an exogenous GlcNAc-1-PT α/βsubunit in a cell.

In an aspect, the disclosure provides method to increase binding of aprotein of interest to cell surface mannose 6-phosphate (Man-6-P)receptors (Man-6-P), by expressing a modified GlcNAc-1-PT α/β subunit ina cell.

In an aspect, the disclosure provides method of enhancingphosphorylation of lysosomal enzymes, by co-expressing a modifiedGlcNAc-1-PT α/β with a lysosomal enzyme of interest such as GBA, GalA,CathD, NPC2, HEXB, GLA, MANBA, alpha-L-idurnoidase, iduronate sulfatase,arylsulfatase B, GAA, or LAMAN in a cell.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F and FIG. 1G depicta schematic, alignment, immunoblots and graphs showing that the spacer-1domain regulates the site of cleavage of the α/β precursor. (FIG. 1A)Schematic of GlcNAc-1-PT α/β subunit modular arrangement and replacementof the human spacer-1 sequence with D. discoideum spacer-1. (FIG. 1B)Immunoblot analysis of WT α/β versus the DS1 deletion mutant expressedin GNPTAB^(−/−) HeLa cells probed with anti-V5 antibody. (FIG. 1C)Phosphotransferase activity toward the simple sugar αMM, using extractsof GNPTAB^(−/−) cells transfected with vector, WT α/β precursor or thevarious mutant cDNAs. Activity was normalized to total proteinconcentration. (FIG. 1D) Inhibition of S1P activity of GNPTAB^(−/−) HeLacells transfected with either WT α/β precursor or the DS1 mutant cDNA.24 h post-transfection, PF-429242 was added to the cells at a finalconcentration of 10 μM and cells were incubated for a further 24 hbefore being harvested. Cell extracts were prepared and 20 μg of eachlysate was separated by SDS-PAGE and subject to western blotting. (FIG.1E) Amino acid alignment of the two GlcNAc-1-PT α subunit S1P substratesites with other known S1P sites. The shaded box shows the conservedconsensus cleavage motif. (FIG. 1F) Immunoblot analysis of the pointmutants, R925A, R879A, and R925A/R879A in the context of either WT α/βor the DS1 deletion mutant. Proteins expressed in GNPTAB^(+/−) HeLacells were separated by SDS-PAGE gel, transferred to nitrocellulose andprobed with anti-V5 antibody. (FIG. 1G) Transfection of GNPTAB^(−/−)HeLa cells with either WT α/β or the various mutants shown in FIG. 1F todetermine enzyme phosphorylation as determined by binding of threeendogenous lysosomal enzymes to CI-MPR-affinity beads. Bound materialwas assayed for activity and values obtained with cells transfected withWT α/β are set to 100%.

FIG. 2A, FIG. 2B and FIG. 2C depict graphs and an immunoblot showingthat the deletion of spacer-1 enhances phosphorylation of severalnon-lysosomal glycoproteins. (FIG. 2A) Mannose phosphorylation of totalsoluble proteins was determined by transfecting GNPTAB^(−/−) HeLa cellswith either vector alone, WT α/β precursor or the DS1 mutant cDNA,followed by [2-³H]mannose labeling. Values shown are calculated as thepercentage of counts recovered with the CI-MPR affinity beads as afraction of the total counts in the phosphotungstic acid precipitate. *represents p=<0.05. (FIG. 2B) GNPTAB^(−/−) HeLa cells wereco-transfected with plasmids encoding either the 3 lysosomal proteins or4 non-lysosomal proteins along with WT α/β precursor or the DS1 mutantcDNA. Cells were labeled with [2-³H]-mannose, followed byimmunoprecipitation of the proteins secreted into the media anddetermination of the percent N-glycans containing Man-6-P. Valuesobtained with WT are set to 1.0. The absolute values of phosphorylationfor the indicated proteins coexpressed with WT α/β precursor were: GLA,36±3%; NPC2, 51±6% C; CathD 25±8%; DNase I, 23±4%; Renin, 21±4%; LIF,24±7%; PoFut2, 12.6%. (FIG. 2C) Western blot of GNPTAB^(−/−) HeLa cellsco-transfected with the expression plasmids for the indicated proteinsalong with empty vector, WT α/β precursor or the DS1 mutant cDNA. Celllysates were incubated with CI-MPR-affinity beads and the binding of thevarious proteins was determined by probing the blot with the followingantibodies: Renin-anti-HA; NPC2, GP, Lamp1 and Lamp2 with antibodiesgenerated against the native protein.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F and FIG. 3G depicta schematic, graphs and immunoblots showing the generation of a minimalenzyme capable of phosphorylating many glycoproteins non-specifically.(FIG. 3A) Schematic of the various α/β precursor deletion constructsexpressed in GNPTAB^(−/−) HeLa cells. (FIG. 3B) Mannose phosphorylationof total soluble proteins was determined by transfecting GNPTAB^(−/−)HeLa cells with WT α/β precursor or the indicated deletion mutant cDNAs,followed by [2-³H]mannose labeling. Values shown are calculated as thepercentage of counts recovered with the CI-MPR affinity beads as afraction of the total counts in the phosphotungstic acid precipitate.The background value of 0.8 ±0.3% was subtracted to yield the finaldepicted vales. * represents p=<0.05 and ** represents p=<0.01. (FIG.3C) Transfection of GNPTAB^(−/−) HeLa cells with either WT α/βprecursor, the N1-D or S1-D deletion mutant cDNAs. The degree ofphosphorylation mediated WT or mutant proteins was determined by bindingof three endogenous lysosomal enzymes to CI-MPR-affinity beads. Boundmaterial was assayed for activity and values obtained with cellstransfected with WT α/β are set to 100%. (FIG. 3D) Western blot of WTα/β precursor and the deletion mutants expressed in GNPTAB^(−/−) HeLacells. The indicated amount of each cell extract was loaded and the α/βprecursor and β subunits were detected with an anti-VS antibody. (FIG.3E) Catalytic activity of WT α/β precursor and the mutants toward αMMusing equal amounts of whole cell extracts. The vector-only transfectedGNPTAB^(−/−) HeLa cell extract served as a control and WT value was setto 100% after subtraction of vector-only background. (FIG. 3F)Immunoblot analysis of GNPTAB^(−/−) HeLa cells co-transfected with theexpression plasmids for the indicated proteins along with empty vector,WT α/β precursor or the indicated deletion mutant cDNAs. Cell lysateswere incubated with CI-MPR-affinity beads and the binding of the variousproteins was determined by probing the blots with the antibodies asdescribed in FIG. 2C, or with anti-myc for PoFut2 and anti-Strep tag forthe vWF A1A2A3 domains. (FIG. 3G) Transfection of GNPTAB^(−/−) HeLacells with either WT α/β precursor, the N1-S3 or S1-S3 deletion mutantcDNAs. The degree of phosphorylation mediated WT or mutant proteins wasdetermined by binding of three endogenous lysosomal enzymes toCI-MPR-affinity beads. Bound material was assayed for activity andvalues obtained with cells transfected with WT α/β are set to 100%.

FIG. 4 depicts a graph showing that phosphorylation of non-lysosomalglycoproteins is mediated by the minimal α/β precursor. GNPTAB^(−/−)HeLa cells were co-transfected with either WT α/β precursor or the S1-S3deletion mutant cDNA, along with expression plasmids for the 4non-lysosomal glycoproteins. 48 h post-transfection, cells were labeledfor 2 h with [2-³H]-mannose, followed by immunoprecipitation of theproteins secreted into the media and determination of the percentN-glycans containing Man-6-P. Values obtained with WT are set to 1. Theabsolute values of phosphorylation for the indicated proteinscoexpressed with WT α/β precursor were: LIF, 26±5%; Renin, 22±2%;PoFut2, 26±2%, DNase I, 22±3%.

FIG. 5 depicts a model for GlcNAc-1-PT function. (Top) GlcNAc-1-PT, inthe basal state, is unable to engage the glycan chains on substratemolecules since the spacer-1 domain (purple) interferes with thecatalytic site formed by the four Stealth domains (pink). Binding of thelysosomal enzyme protein-docking site to the Notch modules/DMAPinteraction domain (orange) induces a conformational change such thatthe spacer-1 domain is now displaced, allowing mannose residues of thelysosomal enzyme high-mannose glycans to enter the catalytic site and bephosphorylated. In some instances, the Man-6-P receptor homology domainof the γ subunit (green) will help guide the oligosaccharides toward thecatalytic site. (Bottom) The minimal enzyme lacking the spacer-1 domainand the Notch modules/DMAP interaction domain is neither inhibited norrequires protein-docking sites on substrate molecules forphosphorylation. This enzyme is expressed at very high levels and ableto phosphorylate all the soluble glycoproteins passing through the Golgiirrespective of their final destination.

FIG. 6 depicts a schematic showing the modular organization of thedifferent domains of human GlcNAc-1-PT α/β precursor and alignment withthe D. discoideum and N. meningitidis GlcNAc-1-PT. It is not certain ifthe D. discoideum protein undergoes proteolytic processing like thehuman protein. The 4 regions shown in green together comprise theStealth, an evolutionarily conserved domain first identified inbacterial proteins involved in capsular polysaccharide biosynthesis.

FIG. 7 depicts a Western blot of HEK 293 cells transfected with eitherWT α/β precursor or the indicated mutant cDNAs. Cell lysates wereincubated with Ni-NTA-agarose to affinity purify the α/β precursor aswell as the β subunit since the various constructs had in addition tothe V-epitope, a 6×-His histidine tag at the C-terminus. The arrowheadindicates the small amount of Q882 cleaved β subunit seen with the WTprotein while the * is the normal β due to cleavage at K928. The R925Amutant also showed a small amount of the Q882-cleavedβ whileK928-cleaved β is completely gone in this case. Both β subunits arecompletely absent with the DS1/R879A/R925A mutant.

FIG. 8A depicts a schematic of GlcNAc-1-PT α/β subunit modulararrangement and replacement of the human spacer-1 sequence with a 26 aalinker sequence comprising of Gly and Ser residues. FIG. 8B depictsimmunoblot analysis of WT α/β versus the DS1 and ΔS1 deletion mutantsexpressed in GNPTAB^(−/−) HeLa cells and probed with anti-V5 antibody.FIG. 8C depicts a graph showing phosphotransferase activity toward thesimple sugar αMM, using extracts of GNPTAB^(−/−) cells transfected withWT α/β precursor or the ΔS1 mutant cDNA. Activity was normalized tototal protein concentration. FIG. 8D depicts an immunoblot showinginhibition of S1P activity of GNPTAB^(−/−) HeLa cells transfected witheither WT α/β precursor, the DS1 or the ΔS1 mutant cDNA. 24 hpost-transfection, PF-429242 was added to the cells at a finalconcentration of 10 μM and cells were incubated for a further 24 hbefore being harvested. Cells extracts were prepared and 20 μg of eachlysate was separated by SDS-PAGE and subject to Western blotting.

FIG. 9 depicts confocal immunofluorescence images of GNPTAB^(−/−) HeLacells transfected with either WT α/β precursor, the DS1 or the ΔS1mutant cDNA, and colocalized with the Golgi markers GOLPH4, respectively(see Methods).

FIG. 10 depicts confocal immunofluorescence images of GNPTAB^(−/−) HeLacells transfected with either WT α/β precursor, or the indicated mutantcDNAs, and colocalized with the Golgi markers GOLPH4, respectively(Methods).

FIG. 11 depicts confocal immunofluorescence images of GNPTAB^(−/−) HeLacells transfected with either WT α/β precursor, the N1-S3 or the S1-S3mutant cDNA, and colocalized with the Golgi markers GOLPH4, respectively(see Methods).

FIG. 12A, FIG. 12B and FIG. 12C depict a schematic and graphs showingthe expression of a minimal GlcNAc-1-phosphotransferase and analysis ofenzyme activity. (FIG. 12A) Schematic of WT GlcNAc-1-phosphotransferaseα/β subunit modular arrangement and that of the minimal enzyme, S1-S3.The minimal enzyme was generated by replacement of the human spacer-1sequence with D. discoideum spacer-1 and removal of amino acids 438-928.(FIG. 12B) Expi293F cells or mouse D9 cells were co-transfected withexpression plasm ids for the indicated lysosomal enzymes along withempty vector, WT α/β precursor or the S1-S3 mutant cDNA. The degree ofphosphorylation mediated by either the WT α/β precursor or the S1-S3mutant was determined for each enzyme by binding to CI-MPR-affinitybeads and assaying the activity of the bound material as described underMethods. Values obtained with empty vector are indicative of activitymediated by the endogenous GlcNAc-1-phosphotransferase. (FIG. 12C)GNPTAB^(−/−) HeLa cells were co-transfected with either WT α/β precursoror the S1-S3 deletion mutant cDNA, along with expression plasmids for 4lysosomal enzymes, while parental HeLa cells were transfected with onlycDNAs for the latter and utilized the endogenousGlcNAc-1-phosphotransferase activity. 48 h post-transfection, cells werelabeled for 2 h with [2-³H]-mannose, followed by immunoprecipitation ofthe proteins secreted into the media and determination of the percentN-glycans containing Man-6-P. The absolute values of the %phosphorylation are shown.

DETAILED DESCRIPTION OF THE INVENTION

The inventors show that a truncated α/β precursor that lacks a number ofthe a subunit elements while retaining the catalytic “Stealth” domainsis expressed at very high levels resulting in a 20-fold greatercatalytic activity than occurs with the WT enzyme. The truncated α/βprecursor stimulated mannose phosphorylation over endogenous levels ofvarious lysosomal and non-lysosomal proteins. Further, the truncatedenzyme increased the formation of glycans with two Man-6-P residueswhich results in much higher affinity for the Manose-6-P receptors.Lysosomal enzyme phosphorylation can be substantially increased byco-transfection with either WT or truncated α/β precursor ofGlcNAc-1-phosphotransferase. The enhanced phosphorylation increasesbinding and uptake by cells. This effect even occurs with lysosomalenzymes such as GalA that are well phosphorylated by the endogenousGlcNAc-1-phosphotransferase. Furthermore, this method enhances thephosphorylation and uptake of LAMAN and GAA, two lysosomal enzymes thatare poorly phosphorylated by endogenous GlcNAc-1-phosphotransferase.Various aspects of the disclosure are described in more detail below.

I. Compositions

In an aspect, the disclosure provides an isolated polypeptide, thepolypeptide comprising GlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/βsubunit. In another aspect, the disclosure provides an isolatedpolynucleotide, the polynucleotide encoding at least one polypeptide,the polypeptide comprising GlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/βsubunit. In still another aspect, the disclosure provides a vectorcomprising a polynucleotide, the polynucleotide encoding at least onepolypeptide, the polypeptide comprising GlcNAc-1-phosphotransferase(GlcNAc-1-PT) α/β subunit. In still yet another aspect, the disclosureprovides a host cell comprising a vector comprising a polynucleotide,the polynucleotide encoding at least one polypeptide, the polypeptidecomprising GlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/β subunit.

In an aspect, a full length GlcNAc-1-PT protein may include 3 subunits,α, β,and γ subunits. The α and β (GlcNAc-1-PT α/β) subunits may be ableto phosphorylate most lysosomal enzymes in the absence of the γ subunit.GlcNAc-1-PT α/β subunits may include various conserved domains. Theconserved domains of the GlcNAc-1-PT α/β subunits may include, arrangedfrom the N- to C-terminus of the polypeptide, a spacer-1 domain(spacer-1), a Notch 1 domain (Notch 1), a Notch 2 domain (Notch 2), aspacer-2 domain (spacer-2), a DNA methyltransferase-associated proteininteraction domain (DMAP), a spacer-3 domain (spacer-3), and a spacer-4domain (spacer-4). The a subunit may include arranged from the N- toC-terminus of the polypeptide a spacer-1, a Notch 1, a Notch 2, aspacer-2, and a DMAP. Spacer-3 may span the α and β subunit, and mayinclude the site at which the α and β subunit may be cleaved, the α/βsubunit cleavage site. The spacer-4 may be in the β subunit.

In an aspect, a GlcNAc-1-PT α/β subunit may be modified by deletion ofone or more conserved domains. By way of non-limiting example, amodified GlcNAc-1-PT α/β subunit may include a deletion of one or moreof spacer-1, Notch 1, Notch 2, spacer-2, DMAP, and a part of spacer-3.In an aspect, a modified GlcNAc-1-PT α/β subunit may include a spacer-1deletion. In an aspect, a modified GlcNAc-1-PT α/β subunit may include aspacer-1 and Notch 1 deletion. In an aspect, a modified GlcNAc-1-PT α/βsubunit may include a spacer-1, and Notch 2 deletion. In an aspect, amodified GlcNAc-1-PT α/β subunit may include a spacer-1, Notch 1, andNotch 2 deletion. In an aspect, a modified GlcNAc-1-PT α/β subunit mayinclude a spacer-1, Notch 1, Notch 2, and spacer-2 deletion. In anaspect, a modified GlcNAc-1-PT α/β subunit may include a spacer-1, Notch1, Notch 2, spacer-2, and DMAP deletion. In an aspect, a modifiedGlcNAc-1-PT α/β subunit may include a spacer-1, Notch 1, Notch 2,spacer-2, and DMAP deletion, and a deletion of a part of spacer-3 at theα/β subunit cleavage site of spacer-3.

In an aspect, the disclosure provides an isolated polypeptide, thepolypeptide comprising GlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/βsubunit, wherein spacer-1 is deleted and the region between Notch 1 andthe α/β cleavage site is deleted. In another aspect, the disclosureprovides an isolated polynucleotide, the polynucleotide encoding atleast one polypeptide, the polypeptide comprisingGlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/β subunit, wherein spacer-1is deleted and the region between Notch 1 and the α/β cleavage site isdeleted. In still another aspect, the disclosure provides a vectorcomprising a polynucleotide, the polynucleotide encoding at least onepolypeptide, the polypeptide comprising GlcNAc-1-phosphotransferase(GlcNAc-1-PT) α/β subunit, wherein spacer-1 is deleted and the regionbetween Notch 1 and the α/β cleavage site is deleted. In still yetanother aspect, the disclosure provides a host cell comprising a vectorcomprising a polynucleotide, the polynucleotide encoding at least onepolypeptide, the polypeptide comprising GlcNAc-1-phosphotransferase(GlcNAc-1-PT) α/β subunit, wherein spacer-1 is deleted and the regionbetween Notch 1 and the α/β cleavage site is deleted.

(a) GlcNAc-1-phosphotransferase

In an aspect, the disclosure provides a GlcNAc-1-phosphotransferase(GlcNAc-1-PT). As used herein, the term “GlcNAc-1-phosphotransferase”includes wild-type GlcNAc-1-phosphotransferase, mutantGlcNAc-1-phosphotransferase, functional homologs ofGlcNAc-1-phosphotransferase and fragments thereof. GlcNAc-1-PT is anα2β2γ2 hexameric protein encoded by two genes. The smaller γ subunit isencoded by the GNPTG gene, whereas the α and β subunits are encoded as asingle α/β precursor by the GNPTAB gene. Proteolytic cleavage of thehuman α/β precursor at K928 is mediated by the Site-1 protease (S1P) inthe Golgi and this cleavage is essential for catalytic competency of theprotein. GlcNAc-1-PT performs the initial and most crucial step in thegeneration of the Man-6-P tag by selectively binding toconformation-dependent protein determinants on lysosomal acid hydrolasesand catalyzing the transfer of GlcNAc-1-P from UDP-GlcNAc to mannoseresidues on high mannose-type N-linked glycans of the hydrolases.Accordingly, a GlcNAc-1-PT of the disclosure, including a functionalhomolog or fragment, generates a Man-6-P tag. In certain embodiments, aGlcNAc-1-P of the disclosure comprises the α/β subunit. The sequenceinformation for the full length human α/β GlcNAc-1-phosphotransferaseamino acid sequence can be found using, for example, the GenBankaccession number CAJ30014.1. The sequence information for the fulllength human α/β GlcNAc-1-phosphotransferase mRNA sequence can be foundusing, for example, the GenBank accession number AM085438.1. In certainembodiments, an α/β GlcNAc-1-phosphotransferase of the disclosurecomprises the sequence set forth in SEQ ID NO:1 (MLFKLLQRQT YTCLSHRYGLYVCFLGVWT IVSAFQFGEV VLEWSRDQYH VLFDSYRDNI AGKSFQNRLC LPMPIDWYTWVNGTDLELL KELQQVREQM EEEQKAMREI LGKNTTEPTK KSEKQLECLL THCIKVPMLVLDPALPANIT LKDLPSLYPS FHSASDIFNV AKPKNPSTNV SVWFDSTKD VEDAHSGLLKGNSRQTVWRG YLTTDKEVPG LVLMQDLAFL SGFPPTFKET NQLKTKLPEN LSSKVKLLQLYSEASVALLK LNNPKDFQEL NKQTKKNMTI DGKELTISPA YLLWDLSAIS QSKQDEDISASRFEDNEELR YSLRSIERHA PWVRNIFIVT NGQIPSWLNL DNPRVTIVTH QDVFRNLSHLPTFSSPAIES HIHRIEGLSQ KFIYLNDDVM FGKDVWPDDF YSHSKGQKVY LTWPVPNCAEGCPGSWIKDG YCDKACNNSA CDWDGGDCSG NSGGSRYIAG GGGTGSIGVG QPWQFGGGINSVSYCNQGCA NSWLADKFCD QACNVLSCGF DAGDCGQDHF HELYKVILLP NQTHYIIPKGECLPYFSFAE VAKRGVEGAY SDNPIIRHAS IANKWKTIHL IMHSGMNATT IHFNLTFQNTNDEEFKMQIT VEVDTREGPK LNSTAQKGYE NLVSPITLLP EAEILFEDIP KEKRFPKFKRHDVNSTRRAQ EEVKIPLVNI SLLPKDAQLS LNTLDLQLEH GDITLKGYNL SKSALLRSFLMNSQHAKIKN QAIITDETND SLVAPQEKQV HKSILPNSLG VSERLQRLTF PAVSVKVNGHDQGQNPPLDL ETTARFRVET HTQKTIGGNV TKEKPPSLIV PLESQMTKEK KITGKEKENSRMEENAENHI GVTEVLLGRK LQHYTDSYLG FLPWEKKKYF QDLLDEEESL KTQLAYFTDSKNTGRQLKDT FADSLRYVNK ILNSKFGFTS RKVPAHMPHM IDRIVMQELQ DMFPEEFDKTSFHKVRHSED MQFAFSYFYY LMSAVQPLNI SQVFDEVDTD QSGVLSDREI RTLATRIHELPLSLQDLTGL EHMLINCSKM LPADITQLNN IPPTQESYYD PNLPPVTKSL VTNCKPVTDKIHKAYKDKNK YRFEIMGEEE IAFKMIRTNV SHVVGQLDDI RKNPRKFVCL NDNIDHNHKDAQTVKAVLRD FYESMFPIPS QFELPREYRN RFLHMHELQE WRAYRDKLKF WTHCVLATLIMFTIFSFFAE QLIALKRKIF PRRRIHKEAS PNRIRV). In other embodiments, aGlcNAc-1-phosphotransferase of the disclosure may have about 80%identity to SEQ ID NO:1, provided it has the same functional activity asGlcNAc-1-PT. For example, a GlcNAc-1-phosphotransferase of thedisclosure may have about 80%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ IDNO:1, provided it has the same functional activity as GlcNAc-1-PT.

In an aspect, in reference to the full length GlcNAc-1-PT (SEQ ID No:1)the spacer-1 is approximately between amino acid 86 and amino acid 322,the Notch 1 and Notch 2 are approximately between amino acid 438 andamino acid 435, the spacer-2 is approximately between amino acid 535 andamino acid 694, DMAP is approximately between amino acid 694 and aminoacid 819, the spacer-3 is approximately between amino acid 819 and aminoacid 955, the α/β subunit cleavage site is approximately at amino acid928, and a spacer-4 is approximately between amino acid 1041 and aminoacid 1149.

It is appreciated that the present directed is directed to homologs ofGlcNAc-1-PT in other organisms and is not limited to the humanGlcNAc-1-PT. Homologs can be found in other species by methods known inthe art. In determining whether a protein has significant homology orshares a certain percentage of sequence identity with a sequence of theinvention, sequence similarity may be determined by conventionalalgorithms, which typically allow introduction of a small number of gapsin order to achieve the best fit. In particular, “percent identity” oftwo polypeptides or two nucleic acid sequences is determined using thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA87:2264-2268, 1993). Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410,1990). BLAST nucleotide searches may be performed with the BLASTNprogram to obtain nucleotide sequences homologous to a nucleic acidmolecule of the invention. Equally, BLAST protein searches may beperformed with the BLASTX program to obtain amino acid sequences thatare homologous to a polypeptide of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., BLASTX and BLASTN) are employed. Seewww.ncbi.nlm.nih.gov for more details.

A GlcNAc-1-PT homolog may be at least 65, 70, 75, 80, 85, 90, or 95%homologous to human GlcNAc-1-PT provided it has the same functionalactivity as GlcNAc-1-PT. In certain embodiments, a GlcNAc-1-PT homologmay be at least 65, 66, 67, 68, 69, or 70% homologous to humanGlcNAc-1-PT provided it has the same functional activity as GlcNAc-1-PT.In different embodiments, a GlcNAc-1-PT homolog may be at least 71, 72,73, 74, 75, 76, 77, 78 or 79% homologous to human GlcNAc-1-PT providedit has the same functional activity as GlcNAc-1-PT. In one embodiment, aGlcNAc-1-PT homolog may be at least 80, 81, 82, 83, 84, 85, 86, 87, 88,or 89% homologous to human GlcNAc-1-PT. In another embodiment, aGlcNAc-1-PT homolog may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100% homologous to GlcNAc-1-PT. In yet another embodiment, aGlcNAc-1-PT homolog may be a truncation or variant that has the samefunctional activity as the full length GlcNAc-1-PT.

In other embodiments, a GlcNAc-1-P of the disclosure comprises the α/βsubunit, wherein spacer-1 is deleted. More specifically, a GlcNAc-1-P ofthe disclosure comprises the α/β subunit, wherein the amino acidsbetween about amino acid 86 to about amino acid 322 are deleted inreference to SEQ ID NO:1. Deletion of spacer-1 gives rise to aGlcNAc-1-P with enhanced ability to phosphorylate a number ofnon-lysosomal glycoproteins that are poorly phosphorylated by thewild-type a GlcNAc-1-P. In still other embodiments, a GlcNAc-1-P of thedisclosure comprises the α/β subunit, wherein the region between Notch 1and the α/β cleavage site is deleted. More specifically, a GlcNAc-1-P ofthe disclosure comprises the α/β subunit, wherein the amino acidsbetween about amino acid 438 and about amino acid 928 are deleted inreference to SEQ ID NO:1. In specific embodiments, a GlcNAc-1-P of thedisclosure comprises the α/β subunit, wherein spacer-1 is deleted andthe region between Notch 1 and the α/β cleavage site is deleted. Morespecifically, a GlcNAc-1-P of the disclosure comprises the α/β subunit,wherein the amino acids between about amino acid 86 to about amino acid322 are deleted and the amino acids between about amino acid 438 andabout amino acid 928 are deleted in reference to SEQ ID NO:1.Importantly, the deletion cannot extend beyond amino acid 928. Removalof spacer-1, together with the region between Notch1 and the α/βcleavage site, results in a GlcNAc-1-P that is reminiscent of thebacterial proteins and cells expressing this minimal GlcNAc-1-PT displaydramatically increased activity toward the simple sugar α-methylD-mannoside (αMM) and non-lysosomal glycoproteins as a consequence ofits high expression level. A GlcNAc-1-PT α/β subunit wherein spacer-1 isdeleted and the region between Notch 1 and the α/β cleavage site isdeleted has about 5-fold greater catalytic activity than wild-typeGlcNAc-1-PT α/β subunit. For example, a GlcNAc-1-PT α/β subunit whereinspacer-1 is deleted and the region between Notch 1 and the α/β cleavagesite is deleted has about 10-fold, about 15-fold, about 20-fold, about25-fold, or about 30 fold greater catalytic activity than wild-typeGlcNAc-1-PT α/β subunit. A GlcNAc-1-PT α/β subunit wherein spacer-1 isdeleted and the region between Notch 1 and the α/β cleavage site isdeleted increases the content of glycans with 2 Man-6-P residuesrelative to wild-type GlcNAc-1-PT α/β subunit. For example, aGlcNAc-1-PT α/β subunit wherein spacer-1 is deleted and the regionbetween Notch 1 and the α/β cleavage site is deleted increases thecontent of glycans with 2 Man-6-P residues by about 1%, about 2%, about3%, about 4%, about 5%, about 10%, about 15%, about 20% or about 25%relative to wild-type GlcNAc-1-PT α/β subunit. Other modifiedGlcNAc-1-Ps of the disclosure are depicted in FIG. 3A.

(b) Enzyme Construct

In an aspect, the present disclosure provides an enzyme construct. Anenzyme construct of the disclosure is a polynucleotide sequence encodingat least one polypeptide, the polypeptide comprising aGlcNAc-1-phosphotransferase or a fragment thereof. As used herein, theterms “polynucleotide sequence of the disclosure” and “enzyme construct”are interchangeable. The present disclosure also provides isolatedpolypeptides encoded by enzyme constructs, vectors comprising enzymeconstructs, and isolated cells comprising said vectors.

i. Polynucleotide Sequence

An enzyme construct of the disclosure is a polynucleotide sequenceencoding at least one polypeptide, the polypeptide comprising aGlcNAc-1-phosphotransferase or a fragment thereof. In certainembodiments, the enzyme construct is a polynucleotide sequence encodingat least one polypeptide, the polypeptide comprisingGlcNAc-1-phosphotransferase α/β subunit. In another embodiment, theenzyme construct is a polynucleotide sequence encoding at least onepolypeptide, the polypeptide comprising GlcNAc-1-phosphotransferase α/βsubunit, wherein spacer-1 is deleted. In still another embodiment, theenzyme construct is a polynucleotide sequence encoding at least onepolypeptide, the polypeptide comprising GlcNAc-1-phosphotransferase α/βsubunit, wherein the region between Notch 1 and the α/β cleavage site isdeleted. In still yet another embodiment, the enzyme construct is apolynucleotide sequence encoding at least one polypeptide, thepolypeptide comprising GlcNAc-1-phosphotransferase α/β subunit, whereinspacer-1 is deleted and the region between Notch 1 and the α/β cleavagesite is deleted. In a different embodiment, the enzyme construct is apolynucleotide sequence encoding at least two polypeptides, thepolypeptides comprising GlcNAc-1-phosphotransferases or fragmentsthereof.

When more than one polypeptide is encoded by a polynucleotide of thedisclosure, the polynucleotide may comprise more than one promotersoperably linked to each polynucleotide encoding a polypeptide. By way ofnon-limiting example, a polynucleotide encoding a polypeptide comprisinga first GlcNAc-1-phosphotransferase or a fragment thereof may beoperably linked to a first promoter and a polynucleotide encoding apolypeptide comprising a second GlcNAc-1-phosphotransferase or afragment thereof may be operably linked to a second promoter. The firstand second GlcNAc-1-phosphotransferase or a fragment thereof may be thesame or different. The first and second promoter may be the same ordifferent. Promoters are described in more detail below.

Alternatively, when more than one polypeptide is encoded by apolynucleotide of the disclosure, the polynucleotide may be operablylinked to a single promoter. In such an embodiment, several strategiescommon in the art may be used to generate more than one expressionproduct. By way of non-limiting example, a splicing signal, internalribosomal entry site (IRES) or proteolytic cleavage site may be insertedbetween the polynucleotides encoding the polypeptides. By way ofnon-limiting example, a polynucleotide encoding a polypeptide comprisinga first GlcNAc-1-phosphotransferase or a fragment thereof and a secondGlcNAc-1-phosphotransferase or a fragment thereof operably linked to asingle promoter may further comprise a splicing signal, IRES orproteolytic cleavage site between the coding regions of the first andsecond GlcNAc-1-phosphotransferase or a fragment thereof.

In each of the above embodiments, “GlcNAc-1-phosphotransferase,” “afragment thereof,” “GlcNAc-1-phosphotransferase α/β subunit,” and“GlcNAc-1-phosphotransferase S1-S3” may be as described in detail abovein Section 1(a), which is hereby incorporated by reference into thissection.

Polynucleotide sequences of the disclosure may be produced from nucleicacids molecules using molecular biological methods known to in the art.Any of the methods known to one skilled in the art for the amplificationof polynucleotide fragments and insertion of polynucleotide fragmentsinto a vector may be used to construct the polynucleotide sequences ofthe disclosure. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (See Sambrook et al.Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory;Current Protocols in Molecular Biology, Eds. Ausubel, et al., GreenePubl. Assoc., Wiley-Interscience, NY).

ii. Vector

In another aspect, the present disclosure provides a vector comprisingan enzyme construct of the disclosure. As used herein, a vector isdefined as a nucleic acid molecule used as a vehicle to transfer geneticmaterial. Vectors include but are not limited to, plasm ids, phasmids,cosmids, transposable elements, viruses (bacteriophage, animal viruses,and plant viruses), and artificial chromosomes (e.g., YACs), such asretroviral vectors (e.g. derived from Moloney murine leukemia virusvectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g.derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectorsincluding replication competent, replication deficient and gutless formsthereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40)vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virusvectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors.

The vector may have a high copy number, an intermediate copy number, ora low copy number. The copy number may be utilized to control theexpression level for the enzyme construct, and as a means to control theexpression vector's stability. In one embodiment, a high copy numbervector may be utilized. A high copy number vector may have at least 31,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies perbacterial cell. In other embodiments, the high copy number vector mayhave at least 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, or 400 copies per host cell. In an alternative embodiment, a lowcopy number vector may be utilized. For example, a low copy numbervector may have one or at least two, three, four, five, six, seven,eight, nine, or ten copies per host cell. In another embodiment, anintermediate copy number vector may be used. For instance, anintermediate copy number vector may have at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copiesper host cell.

Vectors of the present disclosure are typically used for proteinexpression. As is well known in the art, such vectors may possess a widearray of replication origins, multiple cloning sequences, promoters,ribosomal binding sites/ribosome entry sites, translation initiationsites, transcription terminators, etc. Vectors may also contain one ormore polynucleotides sequences encoding for selectable markers,reporters, and peptide tags.

A nucleic acid encoding an enzyme construct may also be operably linkedto a nucleotide sequence encoding a selectable marker. A selectablemarker may be used to efficiently select and identify cells that haveintegrated the exogenous nucleic acids. Selectable markers give the cellreceiving the exogenous nucleic acid a selection advantage, such asresistance towards a certain toxin or antibiotic. Suitable examples ofantibiotic resistance markers include, but are not limited to, thosecoding for proteins that impart resistance to kanamycin, spectomycin,neomycin, gentamycin (G418), ampicillin, tetracycline, chloramphenicol,puromycin, hygromycin, zeocin, and blasticidin.

In some embodiments, the vector may also comprise a transcriptioncassette for expressing reporter proteins. By way of example, reporterproteins may include a fluorescent protein, luciferase, alkalinephosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase,and variants thereof.

An expression vector encoding an enzyme construct may be delivered tothe cell using a viral vector or via a non-viral method of transfer.Viral vectors suitable for introducing nucleic acids into cells includeretroviruses, adenoviruses, adeno-associated viruses, rhabdoviruses, andherpes viruses. Non-viral methods of nucleic acid transfer include nakednucleic acid, liposomes, and protein/nucleic acid conjugates. Anexpression construct encoding an enzyme construct that is introduced tothe cell may be linear or circular, may be single-stranded ordouble-stranded, and may be DNA, RNA, or any modification or combinationthereof.

An expression construct encoding an enzyme construct may be introducedinto the cell by transfection. Methods for transfecting nucleic acidsare well known to persons skilled in the art. Transfection methodsinclude, but are not limited to, viral transduction, cationictransfection, liposome transfection, dendrimer transfection,electroporation, heat shock, nucleofection transfection, magnetofection,nanoparticles, biolistic particle delivery (gene gun), and proprietarytransfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene,jetPEI, Effectene, or DreamFect.

Upon introduction into the cell, an expression construct encoding anenzyme construct may be integrated into a chromosome. In someembodiments, integration of the expression construct encoding an enzymeconstruct into a cellular chromosome may be achieved with a mobileelement. The mobile element may be a transposon or a retroelement. Avariety of transposons are suitable for use in the invention. Examplesof DNA transposons that may be used include the Mu transposon, the Pelement transposons from Drosophila, and members of the Tc1/Marinersuperfamily of transposons such as the sleeping beauty transposon fromfish. A variety of retroelements are suitable for use in the inventionand include LTR-containing retrotransposons and non-LTRretrotransposons. Non-limiting examples of retrotransposons includeCopia and gypsy from Drosophila melanogaster, the Ty elements fromSaccharomyces cerevisiae, the long interspersed elements (LINEs), andthe short interspersed elements (SINEs) from eukaryotes. Suitableexamples of LINEs include L1 from mammals and R2Bm from silkworm.

Integration of the exogenous nucleic acid into a cellular chromosome mayalso be mediated by a virus. Viruses that integrate nucleic acids into achromosome include bacteriophages, adeno-associated viruses andretroviruses. Adeno-associated virus (AAV) vectors may be from human ornonhuman primate AAV serotypes and variants thereof. Suitableadeno-associated viruses include AAV type 1, AAV type 2, AAV type 3, AAVtype 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAVtype 10, and AAV type 11. A variety of retroviruses are suitable for usein the invention. Retroviral vectors may either be replication-competentor replication-defective. The retroviral vector may be analpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus,an epsilonretrovirus, a lentivirus, or a spumaretrovirus. In anembodiment, the retroviral vector may be a lentiviral vector. Thelentiviral vector may be derived from human, simian, feline, equine,bovine, or lentiviruses that infect other mammalian species.Non-limiting examples of suitable lentiviruses includes humanimmunodeficiency virus (HIV), simian immunodeficiency virus (SIV),feline immunodeficiency virus (FIV), bovine immunodeficiency virus(BIV), and equine infectious anemia virus (EIAV).

Integration of an expression construct encoding an enzyme construct intoa chromosome of the cell may be random. Alternatively, integration of anexpression construct encoding an enzyme construct may be targeted to aparticular sequence or location of a chromosome. In general, the generalenvironment at the site of integration may affect whether the integratedexpression construct encoding an enzyme construct is expressed, as wellas its level of expression.

Cells transfected with the expression construct encoding an enzymeconstruct generally will be grown under selection to isolate and expandcells in which the nucleic acid has integrated into a chromosome. Cellsin which the expression construct encoding an enzyme construct has beenchromosomally integrated may be maintained by continuous selection withthe selectable marker as described above. The presence and maintenanceof the integrated exogenous nucleic acid sequence may be verified usingstandard techniques known to persons skilled in the art such as Southernblots, amplification of specific nucleic acid sequences using thepolymerase chain reaction (PCR), and/or nucleotide sequencing.

Nucleic acid molecules are inserted into a vector that is able toexpress the fusion polypeptides when introduced into an appropriate hostcell. Appropriate host cells include, but are not limited to, bacterial,yeast, insect, and mammalian cells.

iii. Regulation

In certain aspects, the expression of a polynucleotide sequence of thedisclosure may be regulated. Such regulation may allow control over whenand where an enzyme construct functions.

Expression vectors typically contain one or more of the followingelements: promoters, terminators, ribosomal binding sites/ribosomalentry sites, and translation initiation sites. Such elements may be usedto control the expression of an enzyme construct of the disclosure.Expression of the nucleic acid molecules of the disclosure may beregulated by a second nucleic acid sequence so that the molecule isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of the nucleic acid molecules of the disclosure maybe controlled by any promoter/enhancer element known in the art. Theterm “promoter”, as used herein, may mean a synthetic ornaturally-derived molecule that is capable of conferring, activating orenhancing expression of a nucleic acid. A promoter may be constitutive,inducible/repressible or cell type specific. In certain embodiments, thepromoter may be constitutive. Non-limiting examples of constitutivepromoters for mammalian cells include CMV, UBC, EF1α, SV40, PGK, CAG,CBA/CAGGS/ACTB, CBh, MeCP2, U6 and H1. In other embodiments, thepromoter may be an inducible promoter. The inducible promoter may beselected from the group consisting of: tetracycline, heat shock, steroidhormone, heavy metal, phorbol ester, adenovirus E1A element, interferon,and serum inducible promoters. In different embodiments, the promotermay be cell type specific. For example, cell type specific promoters forneurons (e.g. syapsin), astrocytes (e.g. GFAP), oligodendrocytes (e.g.myelin basic protein), microglia (e.g. CX3CR1), neuroendocrine cells(e.g. chromogranin A), muscle cells (e.g. desmin, Mb), or cardiomyocytes(e.g. alpha myosin heavy-chain promoter) could be used. In an exemplaryembodiment, a promoter may be the Nrl (rod photoreceptor-specific)promoter or the HBB (haemoglobin beta) promoter. A promoter may furthercomprise one or more specific transcriptional regulatory sequences tofurther enhance expression and/or to alter the spatial expression and/ortemporal expression of a nucleic acid. Non-limiting examples of enhancerinclude the CMV enhancer and the SP1 enhancer.

In an embodiment where more than one polypeptide is encoded by apolynucleotide of the disclosure and the polynucleotide comprises morethan one promoters operably linked to each polynucleotide encoding apolypeptide, the promoters may be the same or different. The term“operably linked,” as used herein, means that expression of a nucleicacid sequence is under the control of a promoter with which it isspatially connected. A promoter may be positioned 5′ (upstream) of thenucleic acid sequence under its control. The distance between thepromoter and a nucleic acid sequence to be expressed may beapproximately the same as the distance between that promoter and thenative nucleic acid sequence it controls. As is known in the art,variation in this distance may be accommodated without loss of promoterfunction.

iv. Host Cell

In another aspect, the present disclosure provides a host cellcomprising a vector of the disclosure. The cell may be a prokaryoticcell or a eukaryotic cell. Appropriate cells include, but are notlimited to, bacterial, yeast, fungal, insect, and mammalian cells. Hostcells according to the present disclosure are cells are maintained invitro in substantially pure cultures (i.e. isolated cells). A host cellcomprising a vector of the disclosure may be used for protein expressionand, optionally, purification. Methods for expressing and, optionally,purifying an expressed protein from a host are standard in the art.

In some embodiments, the host cell comprising a vector of the disclosuremay be used to produce a polypeptide encoded by an enzyme construct ofthe disclosure. Generally, production of a polypeptide of the disclosureinvolves transfecting host cells with a vector comprising an enzymeconstruct and then culturing the cells so that they transcribe andtranslate the desired polypeptide. The isolated host cells may then belysed to extract the expressed polypeptide for subsequent purification.

In some embodiments, the host cell is a prokaryotic cell. Non-limitingexamples of suitable prokaryotic cells include E. coli and otherEnterobacteriaceae, Escherichia sp., Campylobacter sp., Wolinella sp.,Desulfovibrio sp. Vibrio sp., Pseudomonas sp. Bacillus sp., Listeriasp., Staphylococcus sp., Streptococcus sp., Peptostreptococcus sp.,Megasphaera sp., Pectinatus sp., Selenomonas sp., Zymophilus sp.,Actinomyces sp., Arthrobacter sp., Frankia sp., Micromonospora sp.,Nocardia sp., Propionibacterium sp., Streptomyces sp., Lactobacillussp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Acetobacteriumsp., Eubacterium sp., Heliobacterium sp., Heliospirillum sp., Sporomusasp., Spiroplasma sp., Ureaplasma sp., Erysipelothrix sp.,Corynebacterium sp. Enterococcus sp., Clostridium sp., Mycoplasma sp.,Mycobacterium sp., Actinobacteria sp., Salmonella sp., Shigella sp.,Moraxella sp., Helicobacter sp, Stenotrophomonas sp., Micrococcus sp.,Neisseria sp., Bdellovibrio sp., Hemophilus sp., Klebsiella sp., Proteusmirabilis, Enterobacter cloacae, Serratia sp., Citrobacter sp., Proteussp., Serratia sp., Yersinia sp., Acinetobacter sp., Actinobacillus sp.Bordetella sp., Brucella sp., Capnocytophaga sp., Cardiobacterium sp.,Eikenella sp., Francisella sp., Haemophilus sp., Kingella sp.,Pasteurella sp., Flavobacterium sp. Xanthomonas sp., Burkholderia sp.,Aeromonas sp., Plesiomonas sp., Legionella sp. and alpha-proteobaeteriasuch as Wolbachia sp., cyanobacteria, spirochaetes, green sulfur andgreen non-sulfur bacteria, Gram-negative cocci, Gram negative bacilliwhich are fastidious, Enterobacteriaceae-glucose-fermentinggram-negative bacilli, Gram negative bacilli-non-glucose fermenters,Gram negative bacilli-glucose fermenting, oxidase positive. Particularlyuseful bacterial host cells for protein expression include Gram negativebacteria, such as Escherichia coli, Pseudomonas fluorescens, Pseudomonashaloplanctis, Pseudomonas putida AC10, Pseudomonas pseudoflava,Bartonella henselae, Pseudomonas syringae, Caulobacter crescentus,Zymomonas mobilis, Rhizobium meliloti, Myxococcus xanthus and Grampositive bacteria such as Bacillus subtilis, Corynebacterium,Streptococcus cremoris, Streptococcus lividans, and Streptomyceslividans. E. coli is one of the most widely used expression hosts.Accordingly, the techniques for overexpression in E. coli are welldeveloped and readily available to one of skill in the art. Further,Pseudomonas fluorescens, is commonly used for high level production ofrecombinant proteins (i.e. for the development bio-therapeutics andvaccines).

In some embodiments, a host cell is a yeast or fungal cell. Particularlyuseful fungal host cells for protein expression include Asperigillisoryzae, Aspergiffis niger, Trichoderma reesei, Aspergillus nidulans,Fusarium graminearum. Particularly useful yeast host cells for proteinexpression include Candida albicans, Candida maltose, Hansenulapolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichiaguillerimondii, Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Yarrowia lipolytica.

In some embodiments, a host cell is a mammalian cell. Particularlyuseful mammalian host cells for protein expression include Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. HepG2), human embryonic kidney cells, Bos primigenius, and Mus musculus. Ina specific embodiment, the host cells are CHO cells. Additionally, themammalian host cell may be an established, commercially-available cellline (e.g., American Type Culture Collection (ATCC), Manassas, Va.). Thehost cell may be an immortalized cell. Alternatively, the host cell maybe a primary cell. “Primary cells” are cells taken directly from livingtissue (i.e. biopsy material) and established for growth in vitro, thathave undergone very few population doublings and are therefore morerepresentative of the main functional components and characteristics oftissues from which they are derived from, in comparison to continuoustumorigenic or artificially immortalized cell lines.

In an aspect, the host cell has been engineered to produce high levelsof a protein of interest. For example, the host cell has been engineeredto produce a protein that would benefit from being tagged withmannose-6-phosphate (Man-6-P). In certain embodiments, the protein ofinterest is a lysosomal protein. Non-limiting examples of lysosomalproteins include β-glucocebrosidase (GBA), GalA, Cathepsin D (CathD),Niemann-Pick disease type C2 (NPC2), β-hexosaminidase (HEXB),α-Galactosidase (GLA), β-Mannosidase (MANBA), alpha-L-idurnoidase,iduronate sulfatase, arylsulfatase B, acid α-glucosidase (GAA), andlysosomal acid α-mannosidase (LAMAN). Specifically, the lysosomalprotein is acid α-glucosidase (GAA) or lysosomal acid α-mannosidase(LAMAN). These proteins are especially useful in combination with thedisclosed GlcNAc-1-PT as they may be poorly phosphorylated withendogenous GlcNAc-1-PT. In other embodiments, the protein of interest isa non-lysosomal protein. Non-limiting examples of non-lysosomal proteinsinclude DNase1, Renin, leukemia inhibitory factor (LIF), proteinO-fucosyltransferase 2 (PoFUT2), glycopepsinogen (GP), and the vonWillebrand factor A1A2A3 domains.

v. Polypeptide Sequence

In another aspect, the present disclosure provides one or more isolatedpolypeptide(s) encoded by a polynucleotide sequence of the disclsoure.Polynucleotide sequences of the disclosure are described in detail inSection I(b)i, and are hereby incorporated by reference into thissection. As used herein, the term “isolated polypeptide” refers to apolypeptide that has been partially or completely purified from the cellfrom which it was produced. Isolated polypeptides of the disclosure maybe produced using molecular biological methods known to in the art.Generally speaking, a polynucleotide sequence encoding the polypeptideis inserted into a vector that is able to express the polypeptide whenintroduced into an appropriate host cell. Appropriate vectors and hostcells are described in Section 1(b)iii and Section 1(b)iv, respectively.Once expressed, polypeptides may be obtained from cells using commonpurification methods. For example, if the polypeptide has a secretionsignal, expressed polypeptides may be isolated from cell culturesupernatant. Alternatively, polypeptides lacking a secretion signal maybe purified from inclusion bodies and/or cell extract. Polypeptides ofthe disclosure may be isolated from culture supernatant, inclusionbodies or cell extract using any methods known to one of skill in theart, including for example, by chromatography (e.g., ion exchange,affinity, particularly by affinity for the specific antigen afterProtein A, and sizing column chromatography), centrifugation,differential solubility, e.g. ammonium sulfate precipitation, or by anyother standard technique for the purification of proteins; see, e.g.,Scopes, “Protein Purification”, Springer Verlag, N.Y. (1982). Isolationof polypeptides is greatly aided when the polypeptide comprises affinitytag or purification tag, as described herein.

In an embodiment, an isolated polypeptide of the disclosure comprisesGlcNAc-1-phosphotransferase or a fragment thereof. In anotherembodiment, an isolated polypeptide of the disclosure comprisesGlcNAc-1-phosphotransferase α/β subunit. In still another embodiment, anisolated polypeptide of the disclosure comprisesGlcNAc-1-phosphotransferase α/β subunit, wherein spacer-1 is deleted. Instill yet another embodiment, an isolated polypeptide polypeptide of thedisclosure comprises GlcNAc-1-phosphotransferase α/β subunit, whereinthe region between Notch 1 and the α/β cleavage site is deleted. Instill yet another embodiment, an isolated polypeptide of the disclosurecomprises GlcNAc-1-phosphotransferase α/β subunit, wherein spacer-1 isdeleted and the region between Notch 1 and the α/β cleavage site isdeleted.

II. Methods

In an aspect, the disclosure provides a method to increaseoligosaccharide phosphorylation of a protein of interest, the methodcomprising expressing an exogenous GlcNAc-1-PT in a cell. The exogenousGlcNAc-1-PT may be as described in Section I(a). The cell may be a hostcell as described in Section I(b)iv. Specifically, the cell is a CHOcell. The amount of phosphorylation may be increased by greater than 1%relative to phosphorylation in the presence of endogenous GlcNAc-1-PTonly. Additionally, the amount of phosphorylation may be increased bygreater than 2%, greater than 3%, greater than 4%, greater than 5%,greater than 10%, greater than 15%, greater than 20%, greater than 25%,greater than 30%, greater than 35%, greater than 40%, greater than 45%,greater than 50%, greater than 55%, greater than 60%, greater than 65%,greater than 70%, or greater than 75% relative to phosphorylation in thepresence of endogenous GlcNAc-1-PT only. Specifically, when theexogenous GlcNAc-1-PT is GlcNAc-1-phosphotransferase α/β subunit, theamount of phosphorylation may be increased by greater than 2%, greaterthan 3%, greater than 4%, greater than 5%, greater than 10%, greaterthan 15%, greater than 20%, or greater than 25% relative tophosphorylation in the presence of endogenous GlcNAc-1-PT only. Further,when the exogenous GlcNAc-1-PT is GlcNAc-1-phosphotransferase α/βsubunit, wherein spacer-1 is deleted and the region between Notch 1 andthe α/β cleavage site is deleted, the amount of phosphorylation may beincreased by greater than 5%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, greater than 30%, greater than 35%,greater than 40%, greater than 45%, greater than 50%, greater than 55%,greater than 60%, greater than 65%, greater than 70%, or greater than75% relative to phosphorylation in the presence of endogenousGlcNAc-1-PT only. Further, the method may increase the content ofglycans with 2 Man-6-P residues. For example, a GlcNAc-1-PT α/β subunitwherein spacer-1 is deleted and the region between Notch 1 and the α/βcleavage site is deleted increases the content of glycans with 2 Man-6-Presidues by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%,about 15%, about 20% or about 25% relative to wild-type GlcNAc-1-PT α/βsubunit. The protein of interest is a protein that would benefit frombeing tagged with mannose-6-phosphate (Man-6-P). In certain embodiments,the protein of interest is a lysosomal protein. Non-limiting examples oflysosomal proteins include β-glucocebrosidase (GBA), GalA, Cathepsin D(CathD), Niemann-Pick disease type C2 (NPC2), β-hexosaminidase (HEXB),α-Galactosidase (GLA), β-Mannosidase (MANBA), alpha-L-idurnoidase,iduronate sulfatase, arylsulfatase B, acid α-glucosidase (GAA), andlysosomal acid α-mannosidase (LAMAN). Specifically, the lysosomalprotein is acid α-glucosidase (GAA) or lysosomal acid α-mannosidase(LAMAN). In other embodiments, the protein of interest is anon-lysosomal protein. Non-limiting examples of non-lysosomal proteinsinclude DNase1, Renin, leukemia inhibitory factor (LIF), proteinO-fucosyltransferase 2 (PoFUT2), glycopepsinogen (GP), and the vonWillebrand factor A1A2A3 domains.

In another aspect, the disclosure provides a method to increase bindingof a protein of interest to cell surface (Man-6-P)receptors, the methodcomprising expressing an exogenous GlcNAc-1-PT in a cell. The exogenousGlcNAc-1-PT may be as described in Section I(a). The cell may be a hostcell as described in Section I(b)iv. Specifically, the cell is a CHOcell. An increase in binding of a protein of interest to cell surface(Man-6-P) receptors may result in increased protein of interest uptake.The binding may be increased by greater than 1.5-fold relative tophosphorylation in the presence of endogenous GlcNAc-1-PT only.Additionally, the binding may be increased by greater than 2-fold,greater than 3-fold, greater than 4-fold, greater than 5-fold, greaterthan 10-fold, greater than 20-fold, greater than 30-fold, greater than40-fold, greater than 50-fold, greater than 60-fold, greater than70-fold, greater than 80-fold, greater than 90-fold, greater than100-fold, greater than 110-fold, greater than 120-fold, greater than130-fold, greater than 140-fold, or greater than 150-fold relative tobinding in the presence of endogenous GlcNAc-1-PT only. Specifically,when the exogenous GlcNAc-1-PT is GlcNAc-1-phosphotransferase α/βsubunit, the binding may be increased by greater than 1.5-fold, greaterthan 2-fold, greater than 3-fold, greater than 4-fold, greater than5-fold, greater than 10-fold, greater than 20-fold, greater than30-fold, greater than 40-fold, greater than 50-fold, greater than60-fold, greater than 70-fold, greater than 80-fold, greater than90-fold, greater than 100-fold, greater than 110-fold, greater than120-fold, greater than 130-fold, greater than 140-fold, or greater than150-fold relative to binding in the presence of endogenous GlcNAc-1-PTonly. Further, when the exogenous GlcNAc-1-PT isGlcNAc-1-phosphotransferase α/β subunit, wherein spacer-1 is deleted andthe region between Notch 1 and the α/β cleavage site is deleted, thebinding may be increased by greater than 2-fold, greater than 3-fold,greater than 4-fold, greater than 5-fold, greater than 10-fold, greaterthan 20-fold, greater than 30-fold, greater than 40-fold, greater than50-fold, greater than 60-fold, greater than 70-fold, greater than80-fold, greater than 90-fold, greater than 100-fold, greater than110-fold, greater than 120-fold, or greater than 130-fold relative tobinding in the presence of endogenous GlcNAc-1-PT only. The protein ofinterest is a protein that would benefit from being tagged withmannose-6-phosphate (Man-6-P). In certain embodiments, the protein ofinterest is a lysosomal protein. Non-limiting examples of lysosomalproteins include β-glucocebrosidase (GBA), GalA, Cathepsin D (CathD),Niemann-Pick disease type C2 (NPC2), β-hexosaminidase (HEXB),α-Galactosidase (GLA), β-Mannosidase (MANBA), alpha-L-idurnoidase,iduronate sulfatase, arylsulfatase B, acid α-glucosidase (GAA), andlysosomal acid α-mannosidase (LAMAN). Specifically, the lysosomalprotein is acid α-glucosidase (GAA) or lysosomal acid α-mannosidase(LAMAN). In other embodiments, the protein of interest is anon-lysosomal protein. Non-limiting examples of non-lysosomal proteinsinclude DNase1, Renin, leukemia inhibitory factor (LIF), proteinO-fucosyltransferase 2 (PoFUT2), glycopepsinogen (GP), and the vonWillebrand factor A1A2A3 domains.

In various aspects, the method further comprises isolating or purifyingthe protein of interest for use in enzyme replacement therapy. Methodsof isolating or purifying a protein are known in the art. Enzymereplacement therapy (ERT) may be used to treat lysosomal storagediseases. Non-limiting examples of enzymes (and their correspondinglysosomal storage diseases) for use in ERT include glucocerebrosidase(Gaucher disease), α-galactosidase A (Fabry disease), acid α-glucosidase(Pompe disease), alpha-L-idurnoidase (mucopolysaccharidosis I, Hurlersyndrome, Hurler-Scheie syndrome, Scheie syndrome), iduronate sulfatase(mucopolysaccharidosis II, Hunter syndrome), arylsulfatase B(mucopolysaccharidosis VI, Maroteaux-Lamy syndrome). Enzyme replacementtherapy is a lifelong therapy. All products are administeredintravenously either through a peripheral line or central access device.Infusions typically occur once every 2 weeks, or sometimes weekly. Usinga GlcNAc-1-PT of the disclosure, the enzyme prepared may be administeredat lowers doses or less frequent intervals. Further, using a GlcNAc-1-PTof the disclosure, lysosomal enzymes generally not available for use dueto low phosphorylation maybe used for ERT. Further, the production ofGBA containing high levels of Man-6-P offers the opportunity to restoreenzyme activity to cell types in patients with Gaucher disease that lackthe mannose receptor.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Introduction to Examples 1-4

The ability of lysosomes to efficiently degrade intracellular andendocytosed material is dependent upon proper trafficking of the 60 orso soluble acid hydrolases to this organelle. In higher eukaryotes, thesorting process is mediated by the mannose 6-phosphate (Man-6-P)recognition system. Newly synthesized acid hydrolases acquire Man-6-Presidues in the cis-Golgi, which serve as high affinity ligands forbinding to Man-6-P receptors (MPRs) in the trans-Golgi network andsubsequent transport to the endo-lysosomal system (1). The cis-Golgienzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PT) performs theinitial and most crucial step in the generation of the Man-6-P tag byselectively binding to conformation-dependent protein determinants onlysosomal acid hydrolases and catalyzing the transfer of GlcNAc-1-P fromUDP-GlcNAc to mannose residues on high mannose-type N-linked glycans ofthe hydrolases (2). Secretory glycoproteins with identical N-linkedglycans do not acquire the Man-6-P tag as they traverse the secretorypathway. Previous studies from our laboratory have demonstrated rolesfor the two Notch modules and the DNA methyltransferase-associatedprotein (DMAP) interaction domains of GlcNAc-1-PT in the specificrecognition of protein determinants on lysosomal acid hydrolases,resulting in phosphorylation of their high mannose oligosaccharides (3).The likely reason non-lysosomal N-glycosylated proteins are precludedfrom this process and prevented from being incorrectly targeted tolysosomes is their lack of such determinants.

GlcNAc-1-PT is an α2β2γ2 hexameric protein encoded by two genes. Thesmaller γ subunit is encoded by the GNPTG gene, whereas the α and βsubunits are encoded as a single α/β precursor by the GNPTAB gene (4,5). Proteolytic cleavage of the human α/β precursor at K928 is mediatedby the Site-1 protease (S1P) in the Golgi and this cleavage is essentialfor catalytic competency of the protein (6). Besides the Notch and DMAPinteraction domains, the α and β subunits also harbor four Stealthdomains that together form the catalytic core of the protein (FIG. 1A).The Stealth domains of all eukaryotic GlcNAc-1-PTs are highly conservedand resemble sequences within bacterial genes that encodesugar-phosphate transferases involved in cell wall polysaccharidebiosynthesis (FIG. 6) (7). Since the bacterial enzymes transfersugar-phosphates directly to polysaccharide acceptors without theinvolvement of protein determinants, the currently held view is thatmammalian GlcNAc-1-PT, in the course of protein evolution, acquired theNotch and DMAP interaction domains to function in the specificrecognition of protein determinants on lysosomal acid hydrolases.

In addition, GlcNAc-1-PT has four so-called “spacer” domains of whichonly one, spacer-2, has been characterized as the y-subunit binding site(3, 8). Hitherto, no function has been ascribed to the other spacerregions. In this study, we investigated the role of spacer-1 in thefunction of GlcNAc-1-PT. Unexpectedly, we found that spacer-1 dictatescleavage of the α/β precursor precisely at K928 by the site-1 protease(S1P) so as to allow for full catalytic activity since removal ofspacer-1 results in cleavage at an alternate site (Q882) and acatalytically impaired enzyme. In addition, deletion of spacer-1 givesrise to an enzyme with enhanced ability to phosphorylate a number ofnon-lysosomal glycoproteins that are poorly phosphorylated by the WTenzyme. Removal of spacer-1, together with the region between Notch1 andthe α/β cleavage site, results in a minimal enzyme that is reminiscentof the bacterial proteins. Cells expressing this minimal GlcNAc-1-PTdisplay dramatically increased activity toward the simple sugar α-methylD-mannoside (αMM) and non-lysosomal glycoproteins as a consequence ofits high expression level. Together, these findings reveal a novel andunexpected role for spacer-1 in inhibiting phosphorylation ofnon-lysosomal proteins and provide new insight into how GlcNAc-1-PTevolved to specifically phosphorylate lysosomal enzymes while at thesame time excluding non-lysosomal proteins from becoming phosphorylatedand missorted to lysosomes.

Example 1. Deletion of Spacer-1 Results in GlcNAc-1-PT α/β Cleavage atan Alternate Site

In order to analyze the function of the spacer-1 domain of the α/βsubunit of GlcNAc-1-PT, an alignment between the human and D. discoideumGlcNAc-1-PT protein sequence and the bacterialN-acetylglucosamine-1-phosphate transferase sequence was initiallyperformed. As shown in FIG. 6, the human spacer-1 sequence is 200 aalonger than that of the D. discoideum and bacterial proteins, as is thecase with all mammalian GlcNAc-1-PT spacer-1 regions for which sequencedata is available. This suggested that the mammalian spacer-1 regioncould play a unique role not associated with the D. discoideum spacer-1sequence. Hence the 236 aa human spacer-1 sequence was replaced with 29aa of the D. discoideum sequence at the DNA level and the resultingconstruct (FIG. 1A, DS1) was transfected into GNPTAB^(−/−) HeLa cellsgenerated by the CRISPR/Cas9 method (3). Western blot analysis of wholecell extracts expressing the WT and DS1 mutant was performed todetermine if replacement of human spacer-1 with the D. discoideumsequence allowed for efficient folding of the mutant protein and itsexit from the endoplasmic reticulum (ER) to the cis-Golgi where the α/βprecursor is cleaved to the α and β subunits. As shown in FIG. 1B andFIG. 1C, the mutant protein is indeed expressed well, exits the ER, andexhibits 60% of WT catalytic activity toward the simple sugar αMM.However, the bulk of the β subunit product of the proteolytic cleavagemigrated slower on an SDS-PAGE gel than the WT β subunit (FIG. 1B,arrowhead), indicating that most of the DS1 mutant is being cleaved atan alternate site relative to the WT protein which is cleaved at K928(FIG. 1B, *). A small amount of the normal β subunit was also seen withDS1 (FIG. 1B and FIG. 1D, longer exposure, *). This raised the questionas to whether the alternate cleavage resulting from removal of spacer-1is due to the same protease, S1P, that cleaves WT α/β precursor at K928,or if a different protease may be involved. To address this issue, wetreated cells with an inhibitor of S1P, the aminopyrrolidineamidePF-429242 (9). The presence of the inhibitor resulted in loss of the βsubunit formation in both the WT GlcNAc-1-PT and the DS1 mutant (FIG.1D), demonstrating that cleavage at the alternate site is mediated byS1P. If this is the case, an additional consensus S1P cleavage siteshould exist N-terminal to the original cleavage site. An examination ofGlcNAc-1-PT α/β amino acid sequence revealed this to be true with theconsensus key arginine residue, R879, occurring at the invariant −4position, and cleavage postulated to occur at Q882 (FIG. 1E) (10).Cleavage at Q882 is consistent with the increase in molecular mass ofthe β subunit seen with DS1. Mutation of R925 abolishes cleavage of WTGlcNAc-1-PT at K928 (FIG. 1F, lane 2). Mutation of R879, on the otherhand, did not affect the normal processing of the full-length α/βprecursor at K928 (FIG. 1F, lane 3), but abolished cleavage at Q882 forthe DS1 mutant, as shown by loss of the slower migrating β subunit (FIG.1F, lane 6). The trace amount of K928 cleaved β in this case was notaffected (FIG. 1F, lane 6, longer exposure). Mutation of both R925 andR879 resulted in complete loss of β formation (FIG. 1F, lane 7). Thesedata clearly identify Q882 as a novel S1P cleavage site in GlcNAc-1-PTthat is rarely utilized except in the absence of spacer-1 (FIG. 7).

Since the 236 aa human spacer-1 sequence was replaced with the 29 aa D.discoideum sequence, it was possible that utilization of the alternatecleavage site is a consequence of introducing the D. discoideum sequenceas opposed to removal of the human spacer-1 sequence. To exclude thispossibility, another spacer-1 deletion mutant was made in which humanspacer-1 was replaced with a 26 aa linker comprising of the smallresidues Gly and Ser (FIG. 8A, ΔS1). Aspacer-1 (ΔS1) behaved in everyrespect similar to DS1 in that the proteolytic processing mediated byS1P resulted in cleavage for the most part at the new site (Q882) (FIG.8B), and ΔS1 had 40% of WT activity toward αMM (FIG. 8C). Moreover, theS1P inhibitor, PF-429242, blocked formation of the β subunit with ΔS1 asit did with WT and the DS1 mutant of GlcNAc-1-PT (FIG. 8D). Also, bothDS1 and ΔS1 showed identical Golgi localization to WT GlcNAc-1-PT (FIG.9), ruling out mislocalization of these two mutants as a possible causefor the altered cleavage. These results unequivocally show that thepresence of the 236aa spacer-1 sequence in human GlcNAc-1-PT ensurescleavage at K928 instead of Q882.

Example 2. Cleavage at Q882 Results in an Inactive GlcNAc-1-PT

Proteolytic processing of GlcNAc-1-PT α/β precursor in the Golgi atresidue K928 is imperative for a catalytically active enzyme (5, 11).Since there are two S1P cleave sites in α/β, this begs the question asto whether cleavage at the new site instead of at K928 also results inan active enzyme. In order to address this question, the activity of thepoint mutants shown in FIG. 1F toward αMM (FIG. 1C) and a number oflysosomal enzymes, (FIG. 1G) were tested, both in the context of WTGlcNAc-1-PT α/β precursor as well as the DS1 mutant. The variousconstructs were expressed in GNPTAB^(−/−) HeLa cells and 48 hpost-transfection, cells extracts were prepared and an aliquot of eachwas saved to perform the αMM activity assay (FIG. 1C). The remainingextracts were incubated with beads containing immobilizedcation-independent (CI)-MPR to bind the lysosomal enzymes that had beenphosphorylated. The beads were washed and assayed for the extent ofbinding of three lysosmal enzymes as described in Methods (FIG. 1G). Asshown in FIG. 1C and FIG. 1G, the R925A mutant in the context of WT α/βprecursor had only background activity toward both αMM and lysosomalenzymes, in concordance with the prevailing hypothesis that cleavage ofthe α/β precursor is imperative for activity. R879A/WT, on the otherhand, exhibited 30% of the activity toward αMM and between 110-125% ofthe activity toward the three lysosomal enzymes compared to the WT α/βprecursor. When these point mutations were tested individually ortogether in the DS1 background, the various mutants still retained20-30% of WT αMM activity (FIG. 1C). The fact that the R925A/R879A/DS1mutant, which is not proteolytically processed at all, retainedsubstantial activity toward αMM (FIG. 1C) and low levels of activitytoward lysosomal enzymes (FIG. 1G) indicates that uncleaved α/β in theabsence of spacer-1 is partially active. All mutants displayed Golgilocalization identical to WT (FIG. 10). These results show that thecatalytic activity toward lysosomal enzymes associated with DS1 is dueto a combination of the small amount of β originating from cleavage atK928 plus the activity contributed by the α/β precursor, with the majorform of β that is cleaved at Q882 being inactive.

Example 3. Deletion of Spacer-1 Enhances Phosphorylation of SeveralNon-Lysosomal Glycoproteins

The total mannose phosphorylation of soluble glycoproteins inGNPTAB^(−/−) HeLa cells transfected with WT or the DS1 mutant construct,or with vector alone was determined. 48 h post-transfection, the cellswere labeled for 2 h with [2-³H]mannose, and then harvested, washed andlysed in detergent-free buffer followed by ultracentrifugation toseparate membrane proteins from the soluble fractions. The solublefractions were then incubated with immobilized CI-MPR to specificallybind the Man-6-P modified proteins, and then analyzed for their contentof [2-³H]mannose-labeled glycoproteins as described under Methods.Surprisingly, after subtraction of the vector-alone value, the DS1mutant consistently gave a small but statistically significant increasein the level of phosphorylation of total soluble glycoproteins comparedto the WT construct (FIG. 2A). The degree of phosphorylation of thelysosomal proteins, GalA, Cathepsin D (CathD), and Niemann-Pick disease,type C2 (NPC2) by either WT or DS1 was also measured using[2-³H]mannose-labeling, immunoprecipitation and direct glycan analysisas described in Methods. All three lysosomal enzymes showed a similardegree of phosphorylation irrespective of whether WT or the DS1construct was co-transfected into the GNPTAB^(−/−) HeLa cells along withexpression vectors for the individual enzymes (FIG. 2B, left panel, WTvalue set at 1.0). Taken together, these findings raised the possibilitythat the observed increase in total phosphorylation by DS1 was due tophosphorylation of non-lysosomal glycoproteins in addition to lysosomalproteins. In order to determine if this was the case, cDNAs for thenon-lysosomal glycoproteins DNase1, Renin, leukemia inhibitory factor(LIF) and protein O-fucosyltransferase 2 (PoFUT2) were co-transfectedalong either VVTα/β precursor or the DS1 mutant cDNA, and the degree ofphosphorylation quantitated by [2-³H]mannose-labeling. Theseglycoproteins were selected for analysis since they were known toacquire low levels of the Man-6-P tag although they are not lysosomalproteins by nature (12-15). In all four cases, the extent of mannosephosphorylation mediated by DS1 was 1.5-2 fold higher than that achievedwith the WT construct (FIG. 2B, right panel). Consistent with this,Renin but not NPC2 displayed increased binding to immobilized CI-MPRwhen the cDNAs for these two proteins were co-transfected with DS1relative to WT α/β precursor (FIG. 2C). Neither glycopepsinogen (GP) northe membrane glycoproteins, Lamp1 and Lamp2, showed any binding underthese conditions (FIG. 2C). These results show that in the absence ofspacer-1, the phosphorylation mediated by the modified α/β subunits of asubset of non-lysosomal substrates is increased. Together, these datademonstrate that spacer-1 dictates that cleavage of the GlcNAc-1-PT α/βprecursor occurs almost exclusively at K928, and functions to minimizephosphorylation of a number of non-lysosomal glycoproteins.

Example 4. Deletion of Amino Acids 438-926 Results in High LevelExpression of an Active GlcNAc-1-PT

It was previously showed that the two Notch repeats along with the DMAPinteraction domain of the a subunit mediate the selective recognition oflysosomal enzymes (FIG. 3A, WT α/β). Deletion of this region (FIG. 3A,N1-D) dramatically reduced the phosphorylation of total solubleglycoproteins as determined by [2-³H]mannose-labeling (FIG. 3B). Giventhat the majority of proteins phosphorylated by GlcNAc-1-PT are in factlysosomal proteins, this outcome in the absence of the N1-D region isnot surprising. Accordingly, phosphorylation of β-hexosaminidase (HEXB),α-Galactosidase (GLA), and β-Mannosidase (MANBA), as measured by theability to bind to immobilized CI-MPR, was almost completely abrogated(FIG. 3C). In light of the finding that spacer-1 acts as an inhibitorydomain, it was hypothesized that deleting spacer-1 in combination withN1-D (FIG. 3A, S1-D) may partially overcome the inability of GlcNAc-1-PTlacking the two Notch modules and the DMAP interaction domain tophosphorylate lysosomal enzymes. This prediction is borne out by theresults showing a small but statistically significant increase inphosphorylation of total soluble proteins mediated by the S1-D mutantrelative to N1-D (FIG. 3B, compare N1-D vs S1-D), as well as a smallincrease in phosphorylation of HEXB, GLA, and MANBA (14%, 13% and 5%,respectively of WT values) (FIG. 3C). Since the activity of the N1-D andS1-D mutants toward the simple sugar αMM is similar (FIG. 3E, N1-D vsS1-D), these increases in lysosomal enzyme phosphorylation mediated bythe S1-D deletion mutant are best explained by the loss of theinhibitory function of spacer-1.

Additional domains from GlcNAc-1-PT α/β precursor were deleted and stillretain catalytic activity was checked. Deletion of all aa from thebeginning of Notch 1 (C438) through K928, which included the majority ofspacer-3, resulted in a truncated form of GlcNAc-1-PT (FIG. 3A, N1-S3)that was expressed approximately 10-fold greater than WT in theGNPTAB^(−/−) HeLa cells (FIG. 3D, compare lanes 3 and 6), and in spiteof the absence of the two Notch repeats and the DMAP interaction domain,this mutant restored total phosphorylation of soluble glycoproteins toWT levels (FIG. 3B, compare WT vs N1-S3). The mutant did not undergoproteolytic processing since the region deleted extended to the cleavagesite (FIG. 3D), yet it displayed dramatically increased catalyticactivity toward the simple sugar αMM (FIG. 3E). This result showed thatcleavage of the α/β precursor is not a requirement per se for catalyticactivity. Deletions beyond K928 were not tolerated.

The outcome of deleting spacer-1 in the context of N1-S3 was determined.This new construct (FIG. 3A, S1-S3) resulted in a further truncated formof GlcNAc-1-PT α/β precursor that also displayed dramatically enhancedexpression (FIG. 3D, compare lanes 3 and 7) and catalytic activitytoward αMM, at levels similar to N1-S3 (FIG. 3E). Most notably, thetotal phosphorylation of soluble glycoproteins mediated by S1-S3 wasincreased more than 3-fold over WT, whereas N1-S3 was similar to WT(FIG. 3B, compare WT, N1-53 and S1-53). Since the only differencebetween S1-S3 and N1-S3 is the absence of spacer-1, these resultsprovide further evidence for an inhibitory role for spacer-1. Thephosphorylation mediated by S1-S3 of the non-lysosomal proteins Renin,PoFut2, GP, and the von Willebrand factor A1A2A3 domains was alsoincreased relative to N1-S3 (FIG. 3F), as was the case with thelysosomal proteins HEXB, GLA, and MANBA (FIG. 3G). All four mutants(N1-D, S1-D, N1-S3, and S1-S3) localized to the Gogi, similar to WT(FIG. 11).

In order to obtain more quantitative measurements of the degree ofMan-6-P formation mediated by the S1-S3 mutant, [2-³H]mannose-labelingof cells cotransfected with cDNAs for either LIF, Renin, PoFut2, andDNase 1 along with either WT GlcNAc-1-PT or the S1-S3 mutant α/βprecursor cDNA was performed. Coexpression of each of these proteinswith S1-S3 resulted in a 2-4 fold increase in phosphorylation relativeto WT (FIG. 4), in agreement with the markedly enhanced expression andconcomitant increase in activity toward αMM (FIG. 3D and FIG. 3E). Thephosphorylation of these proteins was slightly higher than achieved bythe DS1 mutant (compare FIG. 4 to FIG. 2B).

Discussion for Examples 1-4

The Notch modules and DMAP interaction domain of GlcNAc-1-PT haveessential roles in the selective recognition of lysosomal proteins andphosphorylation of their N-linked glycans (3, 16). Numerous otherglycoproteins that traverse the secretory pathway present very similaror identical N-linked glycans which either do not get phosphorylated oracquire only low levels of the Man-6-P tag (17). The prevailingexplanation for this observation is that unlike lysosomal proteins,non-lysosomal proteins lack the structural determinants that arerecognized and bound by the Notch modules and/or DMAP interaction domainof GlcNAc-1-PT. Thus, the presence of a high mannose oligosaccharide ona protein in itself is insufficient for in vivo phosphorylation of theglycan by GlcNAc-1-PT. In vitro, GlcNAc-1-PT is able to phosphorylatethe simple sugar, αMM, but the Km of the enzyme for this substrate iswell over three orders of magnitude higher than that of a lysosomalenzyme, illustrating the key role of the protein docking sites onlysosomal proteins for GlcNAc-1-PT (18). A key finding of this study isthat in addition to specifically recognizing and phosphorylatinglysosomal proteins, GlcNAc-1-PT contains elements (spacer-1) that serveto prevent phosphorylation of non-lysosomal proteins. This is the firstfunction assigned to the spacer-1 domain. Spacer-1 of human GlcNAc-1-PTis 236 aa long and is highly conserved among vertebrate species. It hasa defined structure (PDB ID:2N6D), consistent with a role other thanjust serving as a “spacer” (19). The spacer-1 region of the lowereukaryote, D. discoideum, on the other hand, is similar in length tothat of N. meningitidis bacterial N-acetylglucosamine-1-phosphatetransferase (FIG. 6) and it is very unlikely that it functions in thesame way as the human sequence. There is no significant identity at theaa level between the human and D. discoideum spacer-1 sequence. Sincethe flanking Stealth 1 & 2 domains are very similar between the twospecies, human GlcNAc-1-PT might tolerate substitution of the humansequence for the D. discoideum sequence. Expression of this chimera inGNPTAB^(−/−) HeLa cells yielded an unexpected result in that DS1, thoughfolded efficiently in the ER and transported to the cis-Golgi just likeWT enzyme, was proteolytically processed differently in the lattercompartment. That it was S1P that mediated this alternate cleavage wasascertained through the use of the S1P inhibitor PF-429242. In a recentstudy analyzing GlcNAc-1-PT patient mutations, Velho et al. reportedthat an in-frame deletion of residues Y937 to M972, resulted in cleavageof the α/β precursor by S1P at an alternate upstream site within the asubunit although the study did not identify the new site (20). Theidentification of Q882 as the alternate cleavage site is in agreementwith the higher molecular mass of the p-subunit seen with DS1. Thereason why S1P cleaves at Q882 rather than at K928 within the a subunitin the absence of spacer-1 is not clear at this point. In light of thefinding that deletion of residues 937-972 also resulted in cleavage atthe new site, one possibility is that spacer-1 interacts with someregion of spacer-3 (aa 819-955, FIG. 1) and thereby influences where S1Pcleaved. An alternate explanation for usage of the new site by S1P inthe absence of spacer-1 is that the steric hindrance normally affordedby this domain to prevent cleavage at Q882 is no longer present,allowing S1P to now cleave primarily at Q882, although a small amount ofprecursor is also cleaved at K928. Interestingly, It was determined thatWT GlcNAc-1-PT yields a trace amount of the catalytically inactiveenzyme as a result of proteolytic processing at Q882. These resultsraise the possibility that vertebrate GlcNAc-1-PT acquired spacer-1 tofacilitate cleavage at K928 and maximize its catalytic efficiency.

In addition to dictating the cleavage site utilized by S1P to generatethe correctly processed form of GlcNAc-1-PT, the results also showspacer-1 to have an important role in minimizing phosphorylation of thehigh mannose glycans of non-lysosomal enzymes. It has been welldocumented that a number of non-lysosomal glycoproteins, including DNaseI, Renin, LIF, and PoFut2 acquire low levels of the Man-6-P tag on theiroligosaccharide chains. While the physiological significance of the lowlevel Man-6-P modification of these proteins is not clear, it seemslikely that extensive phosphorylation of these proteins by GlcNAc-1-PTwould be counter-productive to a cell since the Man-6-P modifiedproteins would be segregated from the secretory pathway for delivery tothe endosomal/lysosomal compartment. The data showing a 1.5-2 foldincrease in the phosphorylation mediated by DS1 over the WT enzyme ofDNase I, Renin, LIF, and PoFut2 indicates a role for spacer-1 ininhibiting phosphorylation of non-lysosomal proteins.

It was previously showed that deletion of the two Notch modules and theDMAP interaction domain (N1-D) virtually abolished the phosphorylationactivity of the mutant GlcNAc-1-PT toward all lysosomal enzymes tested(3). In this regard, it is interesting that the spacer-1 deletion, whencombined with the Notch1-DMAP deletion (S1-D), was able to restore lowlevels of phosphorylation of HEXB, GLA, and MANBA (14%, 13% and 5%,respectively of WT values). Since the N1-S3 mutant does not requireproteolytic processing for catalytic activity, it serves as a goodcontrol for assessing the impact of the spacer-1 deletion in the samecontext. This new construct, S1-S3, which resembles bacterial sugarphosphate transferases (FIG. 6), was expressed at high levels, similarto those obtained with N1-S3, and had similar activity toward αMM.However, the S1-S3 construct increased phosphorylation of total solubleglycoproteins by almost 4-fold over WT, whereas the N1-S3 value wassimilar to WT. The S1-S3-mediated phosphorylation of the lysosomalenzymes HEXB, GLA, and MANBA was also increased compared N1-S3, as wasthe phosphorylation of the non-lysosomal glycoproteins proteinsglycopepsinogen and the vWF A1A2A3 domains. The ability of the S1-S3construct to phosphorylate non-lysosomal proteins that are not actedupon by the WT enzyme indicates that it can function in the absence ofprotein-docking sites, similar to bacterial sugar phosphatetransferases.

Taken together, these findings provide support for the hypothesis thatGlcNAc-1-PT α/β precursor acquired in the course of vertebrate evolutionboth positive (Notch modules and DMAP interaction domain) and negative(spacer-1) regulatory domains that serve to facilitate phosphorylationof lysosomal proteins while simultaneously negating the inherentcapability of the enzyme to phosphorylate non-lysosomal glycoproteins.Based on these findings, we propose the following model to explain howGlcNAc-1-PT functions. In the basal state (FIG. 5A), the spacer-1 domaininterferes with oligosaccharide engagement of the catalytic site formedby the four Stealth domains. Upon binding of the lysosomal enzymeprotein docking site to the Notch modules and DMAP interaction domain, aconformational change occurs such that the spacer-1 domain is displaced,allowing mannose residues of the lysosomal enzyme high-mannose glycansto enter the catalytic site and be phosphorylated. In some instances,the mannose-6-phosphate receptor homology domain of the γ subunit willhelp guide the oligosaccharides toward the catalytic site. Weak-bindingnon-lysosomal glycoproteins such as DNase I may be unable to induce theconformational change required to displace spacer-1, limiting the extentof their phosphorylation. Upon deletion of spacer-1, phosphorylation ofthese proteins is increased. Non-lysosomal glycoproteins that totallylack the structural determinants for binding the Notch modules and DMAPinteraction domain fail to be phosphorylated at all. Removal of spacer-1together with the N1-S3 elements results in an enzyme that is highlyexpressed with full catalytic activity, allowing it to phosphorylate allthe soluble glycoproteins passing through the Golgi (FIG. 5B).

Methods for Examples 1-4

Cell lines—The GNPTAB^(−/−) HeLa cell line has been described in detailelsewhere (3). Cells were maintained in DMEM (Life Technologies)containing 0.11 g/L sodium pyruvate and 4.5 g/L glucose, supplementedwith 10% (vol/vol) FBS (Atlanta Biologicals), 100,000 U/L penicillin,100 mg/L streptomycin (Life Technologies) and 2 mM L-glutamine (LifeTechnologies).

DNA constructs—Human GNPTAB-V5/His in pcDNA6 has been described (11).The various α/β deletion constructs were made by either a 2-stepoverlap-extension (OE) PCR process wherein a PCR-generated restrictionfragment encoding the deletion in question was swapped for the nativecDNA within the same region. In order to generate the DS1 construct, a0.5 kb gBlocks gene fragment was synthesized (IDT Inc.) that encoded theD. discoideum spacer-1 sequence together with the human Stealth1 andStealth 2 sequences and utilized in the first step of the OE-PCR. Pointmutations were generated by the QuikChange site-directed mutagenesismethod and all sequences were confirmed to be correct by DNA sequencing.

The LIF cDNA construct was kindly provided by Richard Steet (Universityof Georgia. Athens, Ga.) while the PoFut2-myc cDNA was a gift fromRobert Haltiwanger (University of Georgia. Athens, Ga.). DNase I,glycopepsinogen, CathD-myc, α-GalA, and NPC2-myc have been described (3,12, 21). Renin-HA cDNA was purchased from Addgene (Cambridge, Mass.),while the plasmid, vWF-A1A2A3-Strep-pCDNA6, was provided by J. EvanSadler (Washington University School of Medicine, St. Louis, Mo.).

Immunofluorescence microscopy—To visualize the subcellular localizationof WT α/β and the various mutants, the different constructs weretransfected into GNPTAB^(−/−) HeLa cells using Lipofectamine 3000 (LifeTechnologies) according to the manufacturer's protocol. 24 hpost-transfection, the cells were fixed and the α/β subunits weredetected with mouse anti-V5 monoclonal antibody (Life Technologies). TheGolgi marker, GOLPH4, was detected with rabbit anti-GOLPH4 polyclonalantibody (Abcam), respectively. The processed cells were mounted inProLong® Gold antifade mounting medium (Life Technologies), and theimages were acquired with either an LSM880 confocal microscope (CarlZeiss Inc.). Images were analyzed by Image J software (Fiji).

Western blotting—Proteins resolved by SDS-PAGE under reducing conditionswere transferred to nitrocellulose membrane and detected with antibodiesas indicated in the figure legends.

[2-³H]Mannose labeling experiments for total solubleglycoproteins—Labeling experiments were performed with transfectedGNPTAB^(−/−) HeLa cells as follows: 48 h post-transfection, cells in6-well plates were incubated with 10 μCi of [2-³H]mannose (Perkin Elmer)for 2 h. Following the 2 h pulse, cells were rinsed twice with PBS andharvested, then resuspended in detergent-free buffer containing 25 mMTris.Cl (pH 7.2) and 150 mM NaCl at 4° C. with a protease inhibitorcocktail (Life Technologies). Cell were lysed by sonication, thensubjected to ultracentrifugation at 100,000×g for 1 h to separate themembrane proteins from the soluble fraction. 100 μl of the solublefraction was then incubated with purified CI-MPR that was covalentlyconjugated to Cyanogen bromide-activated-Sepharose 4B in order to pelletthe mannose-phosphorylated glycoproteins, while 10 μl of the solublefraction was precipitated by 1.5% phosphotungstic acid to obtain total[2-³H]mannose label incorporation into the soluble proteins. This methodallowed for accurate quantification of all the mannose labeledglycoproteins that were phosphorylated by either WT or mutantGlcNAc-1-PT.

[2-³H]Mannose labeling experiments for lysosomal enzymes—Labelingexperiments were performed with transfected GNPTAB^(−/−) HeLa cells asfollows: 48 h post-transfection, cells in 60-mm tissue culture plateswere incubated with 50-150 μCi of [2-³H]mannose (Perkin Elmer) for 2 h,followed by the addition of complete medium containing 5 mM glucose, 5mM mannose and 10 mM NH₄Cl to stop mannose uptake and induce secretion.The cells were incubated for an additional 3 h before the media wascollected. In several experiments, cell extracts were prepared andsubjected to Western blotting for β subunit content to confirm that theconstructs were being expressed at comparable levels.

Immunoprecipitation and oligosaccharide analysis—Acid hydrolasessecreted into the media were immunoprecipitated, and oligosaccharidesisolated and analyzed essentially as described in detail previously(23). For the CathD-myc, NPC2-myc, PoFut2-myc and Renin-HA experiments,20 μl anti-myc monoclonal antibody (Santa Cruz Biotechnology) or 5 μlanti-HA monoclonal antibody (Sigma-Aldrich) was pre-bound to 100 μlProtein G-agarose-PLUS beads (Santa Cruz Biotechnology) prior toimmunoprecipitation of labeled lysosomal hydrolases from the media. Inthe case of GLA, DNase I, and LIF, the secreted enzymes wereimmunoprecipitated with Protein G-agarose-PLUS beads pre-bound toanti-β-Gal antibody (Amicus Therapeutics), and rProteinA-agarose beads(RepliGen) pre-bound to anti-DNase I antibody (Sigma, St. Louis, Mo.),or anti-LIF antibody (generously provided by Frederic Blanchard,University of Nantes, Nantes, France). Immunoprecipitated material wastreated with Endo H (NEB) and filtered with Ultracel-10K (EMDMillipore). The filtrate containing neutral and phosphorylated highmannose glycans was treated with mild acid to remove anyN-acetylglucosamine residues still attached to the phosphate moietiesand applied to a QAE-column matrix to separate the oligosaccharidesbearing zero, one or two Man-6-P residues. The retentate containing EndoH-resistant complex oligosaccharides was treated with Pronase (RocheDiagnostics) and fractionated on ConA-sepharose 4B (GE Healthcare). The[2-³H]-mannose content of each fraction was determined and the percentphosphorylation was calculated as described (23). In all cases, valuesobtained with the mock transfection were subtracted.

References for Examples 1-4

-   1. Kornfeld S (1986) Trafficking of lysosomal enzymes in normal and    disease states. The Journal of clinical investigation 77(1):1-6.-   2. Reitman M L & Kornfeld S (1981) Lysosomal enzyme targeting.    N-Acetylglucosaminylphosphotransferase selectively phosphorylates    native lysosomal enzymes. The Journal of biological chemistry    256(23):11977-11980.-   3. van Meel E, et al. (2016) Multiple Domains of    GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal    Enzymes. The Journal of biological chemistry 291(15):8295-8307.-   4. Bao M, Booth J L, Elmendorf B J, & Canfield W M (1996) Bovine    UDP-N-acetylglucosam ine:lysosomal-enzyme    N-acetylglucosamine-1-phosphotransferase. I. Purification and    subunit structure. The Journal of biological chemistry    271(49):31437-31445.-   5. Kudo M, et al. (2005) The alpha- and beta-subunits of the human    UDP-N-acetylglucosamine:lysosomal enzyme    N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by    a single cDNA. The Journal of biological chemistry    280(43):36141-36149.-   6. Marschner K, Kollmann K, Schweizer M, Braulke T, & Pohl S (2011)    A key enzyme in the biogenesis of lysosomes is a protease that    regulates cholesterol metabolism. Science 333(6038):87-90.-   7. Sperisen P, Schmid C D, Bucher P, & Zilian O (2005) Stealth    proteins: in silico identification of a novel protein family    rendering bacterial pathogens invisible to host immune defense. PLoS    computational biology 1(6):e63.-   8. De Pace R, et al. (2015) Subunit interactions of the    disease-related hexameric GlcNAc-1-phosphotransferase complex. Human    molecular genetics 24(23):6826-6835.-   9. Hay B A, et al. (2007) Am inopyrrolidineam ide inhibitors of    site-1 protease. Bioorganic & medicinal chemistry letters    17(16):4411-4414.-   10. Elagoz A, Benjannet S, Mammarbassi A, Wickham L, & Seidah N    G (2002) Biosynthesis and cellular trafficking of the convertase    SKI-1/S1P: ectodomain shedding requires SKI-1 activity. The Journal    of biological chemistry 277(13):11265-11275.-   11. Qian V, et al. (2015) Analysis of Mucolipidosis II/III GNPTAB    Missense Mutations Identifies Domains of UDP-GlcNAc:lysosomal Enzyme    GlcNAc-1-phosphotransferase Involved in Catalytic Function and    Lysosomal Enzyme Recognition. The Journal of biological chemistry    290(5):30453056.-   12. Nishikawa A, Nanda A, Gregory W, Frenz J, & Kornfeld S (1999)    Identification of amino acids that modulate mannose phosphorylation    of mouse DNase I, a secretory glycoprotein. The Journal of    biological chemistry 274(27):19309-19315.-   13. Blanchard F, et al. (1998) The mannose 6-phosphate/insulin-like    growth factor II receptor is a nanomolar affinity receptor for    glycosylated human leukemia inhibitory factor. The Journal of    biological chemistry 273(33):20886-20893.-   14. Faust P L, Chirgwin J M, & Kornfeld S (1987) Renin, a secretory    glycoprotein, acquires phosphomannosyl residues. The Journal of cell    biology 105(5):1947-1955.-   15. Sleat D E, Zheng H, Qian M, & Lobel P (2006) Identification of    sites of mannose 6-phosphorylation on lysosomal proteins. Molecular    & cellular proteomics: MCP 5(4):686-701.-   16. Qian Y, Flanagan-Steet H, van Meel E, Steet R, & Kornfeld S    A (2013) The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme    N-acetylglucosamine-1-phosphotransferase is a substrate recognition    module. Proceedings of the National Academy of Sciences of the    United States of America 110(25):10246-10251.-   17. Sleat D E, et al. (2013) Extending the mannose 6-phosphate    glycoproteome by high resolution/accuracy mass spectrometry analysis    of control and acid phosphatase 5-deficient mice. Molecular &    cellular proteomics: MCP 12(7):1806-1817.-   18. Lang L, Reitman M, Tang J, Roberts R M, & Kornfeld S (1984)    Lysosomal enzyme phosphorylation. Recognition of a protein-dependent    determinant allows specific phosphorylation of oligosaccharides    present on lysosomal enzymes. The Journal of biological chemistry    259(23):14663-14671.-   19. Serrano P, Geralt, M., Wuthrich, K. (2015) NMR structure of the    140-315 fragment of the N-acetylglucosamine-1-phosphate transferase,    alpha and beta subunits.-   20. Velho R V, et al. (2015) Analyses of disease-related GNPTAB    mutations define a novel GlcNAc-1-phosphotransferase interaction    domain and an alternative site-1 protease cleavage site. Human    molecular genetics 24(12):3497-3505.-   21. Steet R, Lee W S, & Kornfeld S (2005) Identification of the    minimal lysosomal enzyme recognition domain in cathepsin D. The    Journal of biological chemistry 280(39):33318-33323.-   22. Valenzano K J, Remmler J, & Lobel P (1995) Soluble insulin-like    growth factor II/mannose 6-phosphate receptor carries multiple high    molecular weight forms of insulin-like growth factor II in fetal    bovine serum. The Journal of biological chemistry    270(27):16441-16448.-   23. Dustin M L, Baranski T J, Sampath D, & Kornfeld S (1995) A novel    mutagenesis strategy identifies distantly spaced amino acid    sequences that are required for the phosphorylation of both the    oligosaccharides of procathepsin D by N-acetylglucosamine    1-phosphotransferase. The Journal of biological chemistry    270(1):170-179.

Example 5. Method for Producing Highly Phosphylated Lysosomal Enzymesfor Enzyme Replacement Therapy

Enzyme Replacement Therapy (ERT) is currently the major form oftreatment for a number of lysosomal storage diseases, although itsefficacy varies among the individual disorders [1]. Most of theseinherited disorders arise from the lack of activity of a singlelysosomal enzyme which leads to the accumulation of the materialnormally degraded by the enzyme. The buildup of the storage material inthe lysosome eventually results in cell and organ dysfunction. The goalof ERT is to introduce sufficient amounts of normal enzyme into thelysosomes of the defective cells to clear the storage material andrestore lysosome function. This form of therapy was first used inpatients with Type 1 Gaucher disease who lack acid β-glucocebrosidaseactivity and accumulate glucosylceramide primarily in macrophage typecells [2]. The replacement enzyme, containing N-linked glycans withterminal mannose residues, is infused intravenously and taken up bymacrophages via cell surface mannose receptors. The endocytosed enzymeis then transported via endosomes to lysosomes where it functions withgood clinical results in this disorder [3].

Since most cell types lack mannose receptors, the replacement enzymesused to treat lysosomal storage disorders that involve cell types otherthan macrophages utilize binding to mannose 6-phosphate (Man-6-P)receptors at the cell surface for subsequent delivery to lysosomes.These enzymes are purified from the secretions of mammalian cells,mostly Chinese Hamster Ovary cells, engineered to produce high levels ofthe enzyme of interest. This approach is dependent upon the ability ofthe endogenous GlcNAc-1-phosphotransferase to phosphorylate mannoseresidues of the N-glycans of the expressed lysosomal enzyme. Some of thereplacement enzymes produced by this technique are highly phosphorylatedand bind well to the Man-6-P receptors. Others, however, are poorlyphosphorylated, limiting their effectiveness in ERT. This includes thePompe disease enzyme (acid α-glucosidase, GAA) and thealpha-mannosidosis enzyme (lysosomal acid α-mannosidase, LAMAN) [4, 5].

The activity of the endogenous GlcNAc-1-phosphotransferase may beinsufficient to effectively phosphorylate the high levels of GAA andLAMAN being synthesized by the producing cells. To examine thispossibility, Expi293F or cation-independent mannose 6-phosphate receptor(CI-MPR) negative mouse D9 L cells were co-transfected with plasm idsencoding a lysosomal enzyme of interest along with the cDNA for theGlcNAc-1-phosphotransferase α/β precursor. WhileGlcNAc-1-phosphotransferase is an α2β2γ2 hexamer encoded by two genes(GNPTAB and GNPTG), the α/β subunits are able to phosphorylate mostlysosomal enzymes in the absence of γ [6]. In addition, a truncated α/βprecursor (S1-S3) that lacks a number of the a subunit elements whileretaining the catalytic “Stealth” domains was also tested (FIG. 12A).This truncated enzyme is expressed at very high levels resulting in a20-fold greater catalytic activity than occurs with the WT enzyme [7].

The binding of four lysosomal enzymes secreted by the transfected cellsto CI-MPR-beads is shown in FIG. 12B. Increased binding, in this case,reflects a higher degree of phosphorylation of these enzymes. In allinstances, the enzyme secreted by cells co-transfected with thetruncated α/β precursor bound to the CI-MPR-beads to a much greaterextent than observed with enzyme expressed alone in the cells. Theeffect of co-transfection with the WT α/β precursor on the lysosomalenzyme binding to the CI-MPR-beads was variable, ranging from minimalstimulation of GAA binding to 12-fold enhanced binding in the case ofLAMAN. To look more directly at the effects of the α/β precursorconstructs on lysosomal enzyme phosphorylation, HeLa cells with aCRISPR-Cas9 inactivated GNPTAB gene [8] were transfected with thevarious plasmids and then incubated with [2-³H] mannose for 1 hr tolabel the N-linked glycans of the lysosomal enzymes. As a control, theparental HeLa cells with active endogenous GlcNAc-1-phosphotransferasewere transfected with only the plasm ids encoding the lysosomal enzymes.Following a 4 h chase in the presence of NH₄Cl to stimulate secretion ofthe labeled enzyme, the media was harvested and the lysosomal enzyme ofinterest was immunoprecipitated. The immunoprecipitates were thenanalyzed for their content of high mannose N-linked glycans with one ortwo Man-6-P residues [8, 9]. These experiments showed that the truncatedα/β precursor stimulated mannose phosphorylation at various levels overthe parental HeLa cells, depending on the lysosomal enzyme (FIG. 12C).Further, the truncated enzyme increased the formation of glycans withtwo Man-6-P residues (Table 2). This is important since glycans with 2Man-6-P residues bind with much higher affinity to the CI-MPR thanglycans with only 1 Man-6-P residue [10]. The WT α/β precursor alsostimulated Man-6-P formation, but to a lesser extent than observed withthe mutant α/β.

The impact of the increased Man-6-P content of the various lysosomalenzymes on their uptake by HeLa cells is shown in Table 1. With theexception of GAA, the enzymes secreted by cells co-transfected withplasm ids encoding either WT or truncated α/β precursor wereinternalized many fold better than enzyme secreted by cells utilizingonly the endogenous GlcNAc-1-phosphotransferase. Most of the uptake wasblocked by the presence of 5 mM Man-6P in the media, showing that theuptake is mediated by the Man-6-P receptor. The results with LAMAN wereparticularly striking, with Man-6-P-inhibitable uptake being stimulatedby 130- to 153-fold. In the case of GAA, the Man-6-P-dependent uptake ofenzyme secreted by cells co-transfected with the truncated α/β precursorwas 2.6-fold greater than GAA secreted by cells co-transfected with WTα/β precursor or only expressing the endogenousGlcNAc-1-phosphotransferase.

These findings establish that lysosomal enzyme phosphorylation can besubstantially increased by co-transfection with either WT or truncatedα/β precursor of GlcNAc-1-phosphotransferase. The enhancedphosphorylation increases binding to the CI-MPR and uptake by cells.This effect even occurs with lysosomal enzymes such as GalA that arewell phosphorylated by the endogenous GlcNAc-1-phosphotransferase. Butmost important is the finding that this method enhances thephosphorylation and uptake of LAMAN and GAA, two lysosomal enzymes thatare poorly phosphorylated by endogenous GlcNAc-1-phosphotransferase.Enzymes prepared by this method have the potential to significantlyimprove their usefulness in ERT. In addition to providing better celluptake, these preparations may allow lower doses to be administered topatients, perhaps at less frequent intervals. The method should beapplicable to the production of lysosomal enzymes for other lysosomalstorage diseases that may be amenable to ERT. In addition, theproduction of GBA containing high levels of Man-6-P offers theopportunity to restore enzyme activity to cell types in patients withGaucher disease that lack the mannose receptor. This might serve toprovide additional benefit to the current therapy which is directedspecifically to macrophages.

Methods for Example 5

Cell lines—Expi293F cells are from Life Technologies. These cells weregrown in suspension in Expi293 expression medium (Life Technologies).The GNPTAB^(−/−) HeLa cell line has been described in detail elsewhere[8]. Parental and GNPTAB^(−/−) HeLa cell were maintained as a monolayerin DMEM (Life Technologies) containing 0.11 g/L sodium pyruvate and 4.5g/L glucose, supplemented with 10% (vol/vol) FBS (Atlanta Biologicals),100,000 U/L penicillin, 100 mg/L streptomycin (Life Technologies) and 2mM L-glutamine (Life Technologies). CI-MPR negative mouse L-cells (D9cell line) have been described [11]. D9 cells were maintained as amonolayer in α-MEM (Life Technologies) containing 100,000 U/L penicillinand 100 mg/L streptomycin (Life Technologies).

DNA constructs—Human GNPTAB-V5/His and the S1-S3 deletion mutant inpcDNA6 has been described [7]. The LAMAN-myc-Flag cDNA was purchasedfrom Origene while the GAA cDNA was a kind gift of Eline van Meel(Leiden University, The Netherlands). The GBA and GLA cDNAs weregenerously provided by Amicus Therapeutics.

CI-MPR affinity chromatography and enzyme assays—Soluble bovine CI-MPRwas purified from fetal bovine serum and covalently conjugated toCyanogen bromide-activated-Sepharose 4B (Sigma-Aldrich) as described[12]. Media from 2-day transfected Expi293F cells or mouse D9 cells wereincubated with the CI-MPR beads at 4° C. for 1 h to bind thephosphorylated lysosomal enzymes. The beads were then sedimented, washedwith buffer (25 mM Tris-Cl, pH7.2, 150 mM NaCl and 1% Triton-X 100), andassayed for lysosomal enzyme activity as described [13]. The amount ofthe starting enzyme recovered on the beads was calculated.

Cell uptake of lysosomal enzymes—Parental HeLa cells were plated on a12-well plate at around 80% density one day prior to the cell uptakeexperiment. Media containing each enzyme from the producing cells wasadded to the parental HeLa cells in a final volume of 500 μl. Forcompetition experiments, Man-6-P was added to a final concentration of 5mM. Cells were incubated for an additional 24 h, following which mediaand cells were collected separately. Cells were rinsed twice with PBS,then lysed in 25 mM Tris-Cl, pH 7.2, 150 mM NaCl, 1% Triton-X 100, andprotease inhibitor cocktail (Life Technologies). The media and lysedcells were centrifuged at 20,000×g, and the activity of the enzyme inthe supernatant of the media and cell lysate was assayed.

[2-³H]Mannose labeling experiments for lysosomal enzymes—Labelingexperiments were performed with transfected GNPTAB^(−/−) parental HeLacells as follows: 48 h post-transfection, cells in 60-mm tissue cultureplates were incubated with 50-150 μCi of [2-³H]mannose (Perkin Elmer)for 2 h, followed by the addition of complete medium containing 5 mMglucose, 5 mM mannose and 10 mM NH₄Cl to stop mannose uptake and inducesecretion. The cells were incubated for an additional 3 h before themedia was collected for analysis.

Immunoprecipitation and oligosaccharide analysis—Acid hydrolasessecreted into the media were immunoprecipitated, and oligosaccharidesisolated and analyzed essentially as described in detail previously [9].Since the LAMAN, GAA and GBA cDNAs were appended with a myc-tag, 20 μlanti-myc monoclonal antibody (Santa Cruz Biotechnology) was pre-bound to100 μl Protein G-agarose-PLUS beads (Santa Cruz Biotechnology) prior toimmunoprecipitation of labeled lysosomal hydrolases from the media. Inthe case of GLA, the secreted enzyme was immunoprecipitated with ProteinG-agarose-PLUS beads pre-bound to anti-β-Gal antibody (AmicusTherapeutics). Immunoprecipitated material was treated with Endo H (NEB)and filtered with Ultracel-10K (EMD Millipore). The filtrate containingneutral and phosphorylated high mannose glycans was treated with mildacid to remove any N-acetylglucosamine residues still attached to thephosphate moieties and applied to a QAE-column matrix to separate theoligosaccharides bearing zero, one or two Man-6-P residues. Theretentate containing Endo H-resistant complex oligosaccharides wastreated with Pronase (Roche Diagnostics) and fractionated onConA-sepharose 4B (GE Healthcare). The [2-³H]-mannose content of eachfraction was determined and the percent phosphorylation was calculatedas described [9].

TABLE 1 Cell uptake of lysosomal enzymes phosphorylated with endogenousGlcNAc-1- phosphotransferase or overexpressed WT enzyme or the S1-S3mutant. Uptake experiments were performed either in the absence ofMan-6-P, or with 5 mM Man-6-P to competitively inhibit uptake of thephosphorylated enzymes. % uptake* Total U M6PR Fold Enzyme PTase AddedMan-6-P Dependent Increase LAMAN Endogenous 670 − 0.76 ± 0.23 0.14 +0.62 ± 0.27 WT α/β 359 − 24.70 ± 1.34  21.5 153 X    + 3.20 ± 0.77 S1-S3530 − 22.46 ± 4.07  18.2 130 X    +  4.3 ± 1.19 GAA Endogenous 5.7 −4.05 ± 0.46 3.8 + 0.22 ± 0.42 WT α/β 3.8 − 3.31 ± 1.13 2.9 0.8 X + 0.44± 1.18 S1-S3 3.2 − 12.79 ± 0.90  8.8 2.6 X + 2.99 ± 1.59 GBA Endogenous2.7 − 10.74 ± 1.94  5.1 + 5.65 ± 1.51 WT α/β 2.3 − 46.73 ± 4.00  37.47.3 X + 9.37 ± 1.26 S1-S3 2.8 − 40.74 ± 11.53 41.1 8.0 X + 8.87 ± 4.21GLA Endogenous 194 − 2.72 ± 0.91 1.7 + 1.00 ± 0.11 WT α/β 141 − 9.53 ±0.11 8.2 4.8 X + 1.30 ± 0.13 S1-S3 237 − 7.01 ± 0.69 8.1 3.5 X + 0.95 ±0.11 Note: 1U = 1 nmol 4-Methylumbeliiferone released per hour *% uptakeper 200 ug cell protein in 24 hour

TABLE 2 Distribution of high mannose glycans with one or two Man-6-Presidues present on lysosomal enzymes. The data presented in FIG. 12C isfurther broken down to show the content of glycans with 1 or 2 Man-6-Presidues among the lysosomal enzymes acted upon by the endogenousGlcNAc-1-phosphotransferase or the overexpressed WT α/β precursor or theS1-S3 deletion mutant. % Oligosaccharide phosphorylation Enzyme PTaseHM + 1PM HM + 2PM Total LAMAN Endogenous 4.5 1.0 5.5 WT 9.7 3.4 13.1S1-S3 24.3 23.9 48.2 GAA WT 4.2 1.1 5.2 S1-S3 8.3 2.7 11.0 GBA WT 6.41.2 7.6 S1-S3 27.2 9.7 36.9 GLA WT 23.7 4.0 27.8 S1-S3 49.6 12.4 62.0HM—High Mannose oligosaccharide PM—phosphomonoester

References for Example 5

-   1. Lachmann, R. N., Enzyme replacement therapy for lysosomal storage    diseases. Curr Opin Pediatr, 2011. 23(6): p. 588-93.-   2. Barton, N. W., et al., Replacement therapy for inherited enzyme    deficiency—macrophage-targeted glucocerebrosidase for Gaucher's    disease. N Engl J Med, 1991. 324(21): p. 1464-70.-   3. Weinreb, N. J., et al., Long-term clinical outcomes in type 1    Gaucher disease following 10 years of imiglucerase treatment. J    Inherit Metab Dis, 2013. 36(3): p. 543-53.-   4. McVie-Wylie, A. J., et al., Biochemical and pharmacological    characterization of different recombinant acid alpha-glucosidase    preparations evaluated for the treatment of Pompe disease. Mol Genet    Metab, 2008. 94(4): p. 448-55.-   5. Roces, D. P., et al., Efficacy of enzyme replacement therapy in    alpha-mannosidosis mice: a preclinical animal study. Hum Mol    Genet, 2004. 13(18): p. 1979-88.-   6. Qian, Y., et al., Functions of the alpha, beta, and gamma    subunits of UDP-GlcNAc:lysosomal enzyme    N-acetylglucosamine-1-phosphotransferase. J Biol Chem, 2010.    285(5): p. 3360-70.-   7. Lin Liu, W.-S. L., Balraj Doray, and Stuart Kornfeld, Regulation    of Lysosmal Enzyme Phosphorylation: Role of the Spacer-1 Domain of    GlcNAc-1-Phosphotransferase. manuscript in preparation.-   8. van Meel, E., et al., Multiple Domains of    GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal    Enzymes. J Biol Chem, 2016. 291(15): p. 8295-307.-   9. Dustin, M. L., et al., A novel mutagenesis strategy identifies    distantly spaced amino acid sequences that are required for the    phosphorylation of both the oligosaccharides of procathepsin D by    N-acetylglucosamine 1-phosphotransferase. J Biol Chem, 1995.    270(1): p. 170-9.-   10. Tong, P. Y., W. Gregory, and S. Kornfeld, Ligand interactions of    the cation-independent mannose 6-phosphate receptor. The    stoichiometry of mannose 6-phosphate binding. J Biol Chem, 1989.    264(14): p. 7962-9.-   11. Gabel, C. A. and S. A. Foster, Lysosomal enzyme trafficking in    mannose 6-phosphate receptor-positive mouse L-cells: demonstration    of a steady state accumulation of phosphorylated acid hydrolases. J    Cell Biol, 1986. 102(3): p. 943-50.-   12. Valenzano, K. J., J. Remmler, and P. Lobel, Soluble insulin-like    growth factor II/mannose 6-phosphate receptor carries multiple high    molecular weight forms of insulin-like growth factor II in fetal    bovine serum. J Biol Chem, 1995. 270(27): p. 16441-8.-   13. Qian, Y., et al., Analysis of Mucolipidosis II/III GNPTAB    Missense Mutations Identifies Domains of UDP-GlcNAc:lysosomal Enzyme    GlcNAc-1-phosphotransferase Involved in Cataytic Function and    Lysosomal Enzyme Recognition. J Biol Chem, 2015. 290(5): p. 3045-56.

1. A modified GlcNAc-1-phosphotransferase (GlcNAc-1-PT) α/β subunitcomprising internal deletion of amino acids with reference tofull-length human GlcNAc-1-PT α/β subunit having the sequence of SEQ IDNO:1, wherein SEQ ID NO:1 comprises from N to C-terminus, a spacer-1domain (spacer-1), a Notch 1 domain (Notch 1), a Notch 2 domain (Notch2), a spacer-2 domain, a DNA methyltransferase-associated proteininteraction domain (DMAP), a spacer-3 domain (spacer-3), a α/β subunitcleavage site, and a spacer-4 domain (spacer-4), wherein the spacer-1 isinternally deleted.
 2. The modified GlcNAc-1-PT α/β subunit of claim 1,wherein the amino acids between amino acid 86 to amino acid 322 aredeleted, with reference to SEQ ID NO:1.
 3. The modified GlcNAc-1-PT α/βsubunit of claim 1, wherein the region between Notch 1 and the α/βcleavage site is deleted.
 4. The modified GlcNAc-1-PT α/β subunit ofclaim 1, wherein the amino acids between amino acid 438 and amino acid928 are deleted, with reference to SEQ ID NO:1.
 5. The modifiedGlcNAc-1-PT α/β subunit of claim 1, wherein spacer-1 is deleted and theregion between Notch 1 and the α/β cleavage site is deleted.
 6. Themodified GlcNAc-1-PT α/β subunit of claim 1, wherein the amino acidsbetween amino acid 86 to amino acid 322 are deleted and the amino acidsbetween amino acid 438 and amino acid 928 are deleted, with reference toSEQ ID NO:1.
 7. The modified GlcNAc-1-PT α/β subunit of claim 4, whereinthe deletion between Notch 1 and the α/β cleavage site does not extendbeyond amino acid 928 with reference to SEQ ID NO:1.
 8. A vectorcomprising the polynucleotide of claim
 1. 9. A host cell comprising thevector of claim 8, wherein the host cell is engineered to produce highlevels of a protein of interest. 10.-13. (canceled)
 14. The host cell ofclaim 9, wherein the protein of interest is a lysosomal protein isselected from the group consisting of β-glucocebrosidase (GBA),Cathepsin D (CathD), Niemann-Pick disease type C2 (NPC2),β-hexosaminidase (HEXB), α-Galactosidase (GLA), β-Mannosidase (MANBA),alpha-L-iduronidase, iduronate sulfatase, arylsulfatase B, acidα-glucosidase (GAA), alpha-N-Acetylducosaminidase (NAGlu), and lysosomalacid α-mannosidase (LAMAN).
 15. (canceled)
 16. The host cell of claim 9,wherein the protein of interest is a non-lysosomal protein is selectedfrom the group consisting of DNase1, Renin, leukemia inhibitory factor(LIF), protein O-fucosyltransferase 2 (PoFUT2), glycopepsinogen (GP),and the von Willebrand factor A1A2A3 domains.
 17. The host cell of claim9, wherein the protein of interest is selected from the group consistingof the Pompe disease enzyme (acid α-glucosidase, GAA),alpha-N-Acetylducosaminidase (NAGlu), and the alpha-mannosidosis enzyme(lysosomal acid α-mannosidase, LAMAN).
 18. A method to increaseoligosaccharide phosphorylation of a protein of interest, the methodcomprising expressing an exogenous modified GlcNAc-1-PT α/β subunit in acell. 19.-27. (canceled)
 28. The method of claim 18, wherein theGlcNAc-1-PT α/β subunit comprises SEQ ID NO:1, wherein SEQ ID NO:1comprises from N to C-terminus, a spacer-1 domain (spacer-1), a Notch 1domain (Notch 1), a Notch 2 domain (Notch 2), a spacer-2 domain, a DNAmethyltransferase-associated protein interaction domain (DMAP), aspacer-3 domain (spacer-3), a α/β subunit cleavage site, and a spacer-4domain (spacer-4).
 29. The method of claim 28, wherein the spacer-1 isinternally deleted.
 30. The method of claim 28, wherein the amino acidsbetween amino acid 86 to amino acid 322 are deleted with reference toSEQ ID NO:1.
 31. The method of claim 28, wherein the region betweenNotch 1 and the α/β cleavage site is deleted.
 32. The method of claim28, wherein the amino acids between amino acid 438 and amino acid 928are deleted with reference to SEQ ID NO:1.
 33. The method of claim 28,wherein spacer-1 is deleted and the region between Notch 1 and the α/βcleavage site is deleted.
 34. The method of claim 28, wherein the aminoacids between amino acid 86 to amino acid 322 are deleted and the aminoacids between amino acid 438 and amino acid 928 are deleted withreference to SEQ ID NO:1. 35.-38. (canceled)